Fully Human Antibodies That Bind to Vascular Endothelial Growth Factor Receptor (VEGFR2)

ABSTRACT

There is disclosed compositions and methods relating to anti-VEGFR2 antibodies. More specifically, there is disclosed fully human antibodies that bind VEGFR2, VEGFR2-binding fragments and derivatives of such antibodies, and VEGFR2-binding polypeptides comprising such fragments. Further still, there is disclosed antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating or diagnosing subjects having various cancers.

CROSS REFERENCE TO RELATED APPLICATION

The present patent application is a divisional of U.S. patentapplication Ser. No. 14/680,865, filed Apr. 7, 2015, which is acontinuation of U.S. patent application Ser. No. 13/854,071, filed Mar.30, 2013, now U.S. Pat. No. 9,029,510, which claims priority to U.S.Patent Application 61/618,658 filed 30 Mar. 2012. The contents of all ofthese applications are hereby incorporated by reference herein in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 29, 2021, isnamed 2021-07-29_01223-0033-02US_Seq_Listing.txt and is 84 kilobytes insize.

TECHNICAL FIELD

The present disclosure provides compositions and methods for fully humananti-VEGFR2 antibodies. More specifically, the present disclosureprovides human antibodies that bind VEGFR2, VEGFR2-binding fragments andderivatives of such antibodies, and VEGFR2-binding polypeptidescomprising such fragments. Further still, the present disclosureprovides antibody fragments and derivatives and polypeptides, andmethods of using such antibodies, antibody fragments and derivatives andpolypeptides, including methods of treating or diagnosing subjectshaving VEGFR2-related disorders or conditions.

BACKGROUND

Angiogenesis is an important cellular event in which vascularendothelial cells proliferate, prune and reorganize to form new vesselsfrom preexisting vascular networks. There are compelling evidences thatthe development of a vascular supply is essential for normal andpathological proliferative processes (Folkman and Klagsbrun (1987)Science 235:442-447). Delivery of oxygen and nutrients, as well as theremoval of catabolic products, represent rate-limiting steps in themajority of growth processes occurring in multicellular organisms. Thus,it has been generally assumed that the vascular compartment isnecessary, not only for organ development and differentiation duringembryogenesis, but also for wound healing and reproductive functions inthe adult.

Angiogenesis is also implicated in the pathogenesis of a variety ofdisorders, including but not limited to, cancer, proliferativeretinopathies, age-related macular degeneration, rheumatoid arthritis(RA), and psoriasis. Angiogenesis is essential for the growth of mostprimary solid tumors and their subsequent metastasis. Tumors can absorbsufficient nutrients and oxygen by simple diffusion up to a size of 1-2mm, at which point their further growth requires the elaboration ofvascular supply. This process is thought to involve recruitment of theneighboring host mature vasculature to begin sprouting new blood vesselcapillaries, which grow towards, and subsequently infiltrate, the tumormass. In addition, tumor angiogenesis involves the recruitment ofcirculating endothelial precursor cells from the bone marrow to promoteneovascularization Kerbel, Carcinogenesis 21:505-515, 2000; and Lyndenet al., Nat. Med. 7:1194-1201, 2001.

While induction of new blood vessels is considered to be the predominantmode of tumor angiogenesis, some tumors may grow by co-opting existinghost blood vessels. The co-opted vasculature then regresses, leading totumor regression that is eventually reversed by hypoxia-inducedangiogenesis at the tumor margin. Holash et al., Science 284:1994-1998,1999.

In many instances, the process begins with the activation of existingvascular endothelial cells in response to a variety of cytokines andgrowth factors. In cancer, tumor released cytokines or angiogenicfactors stimulate vascular endothelial cells by interacting withspecific cell surface receptors. The activated endothelial cells secreteenzymes that degrade the basement membrane of the vessels, allowinginvasion of the endothelial cells into the tumor tissue. Once situated,the endothelial cells differentiate to form new vessel offshoots ofpre-existing vessels. The new blood vessels provide nutrients to thetumor, facilitating further growth, and also provide a route formetastasis.

Numerous angiogenic factors have been identified, including theparticularly potent factor VEGF. VEGF was initially purified from theconditioned media of folliculostellate cells and from a variety of celllines. Various forms of VEGF bind as high affinity ligands to a suite ofVEGF receptors (VEGFRs). VEGFRs are tyrosine kinase receptors, many ofwhich are important regulators of angiogenesis. The VEGFR familyincludes 3 major subtypes: VEGFR1, VEGFR2 (also known as Kinase InsertDomain Receptor, “KDR”, in humans), and VEGFR3. Among VEGF forms,VEGF-A, VEGF-C and VEGF-D are known to bind and activate VEGFR2.

VEGF, acting through its cognate receptors, can function as anendothelial specific mitogen during angiogenesis. In addition, there issubstantial evidence that VEGF and VEGFRs are up-regulated in conditionscharacterized by inappropriate angiogenesis, such as cancer. As aresult, a great deal of research has focused on the identification oftherapeutics that target and inhibit VEGFs or VEGFRs.

Therapeutic approaches that target or inhibit VEGFs or VEGFRs includeantibodies, peptides, and small molecule kinase inhibitors. Of these,antibodies are widely used for in vivo recognition and inhibition ofligands and cellular receptors. Highly specific antibodies have beenused to block receptor-ligand interaction, thereby neutralizing thebiological activity of the components, and also to specifically delivertoxic agents to cells expressing the cognate receptor on its surface. Asa result, there remains a need for effective therapeutics that canspecifically inhibit VEGF/VEGFR pathways as a treatment for disorderscharacterized by inappropriate angiogenesis, such as cancer.

The anti-VEGF antibody “Bevacizumab (BV)”, also known as “rhuMAb VEGF”or “Avastin®” is a recombinant humanized anti-VEGF monoclonal antibodygenerated according to Presta et al., Cancer Res. 57:4593-4599, 1997. Itcomprises mutated human IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of Bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.Bevacizumab has a molecular mass of about 149,000 daltons and isglycosylated.

SUMMARY

The anti-VEGFR2 binding proteins described herein may be used, forexample, to detect VEGFR2 in vivo or in vitro. Additionally, certainVEGFR2 binding proteins described herein may be used to treat diseasesassociated with VEGFR2-mediated biological activity. For example, VEGFR2mediates the pro-angiogenic effects of VEGF, and accordingly, certainVEGFR2 binding proteins of the disclosure may be used to inhibitangiogenesis in a human patient. Certain VEGFR2 binding proteins of thedisclosure may be used to treat disorders such as cancers, inflammatorydiseases, autoimmune diseases and retinopathies. Many disorders relatedto the hyperproliferation of cells of a tissue will include anangiogenic component, and thus it is expected that certain VEGFR2binding proteins described herein can be used to treat such disorders.

The present disclosure provides a fully human antibody of an IgG classthat binds to a VEGFR2 epitope with a binding affinity of at least10⁻⁶M, that has a heavy chain variable domain sequence that is at least95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ IDNO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45,SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO.55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ IDNO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 73, SEQID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46,SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO.56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ IDNO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80, and combinations thereof.Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2(called VB-A2 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called VB-A3 herein),SEQ ID NO. 5/SEQ ID NO. 6 (called VB-A7 herein), SEQ ID NO. 7/SEQ ID NO.8 (called VBV-A7 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called VB-A9herein), SEQ ID NO. 11/SEQ ID NO. 12 (called VB-A10 herein), SEQ ID NO.13/SEQ ID NO. 14 (called VB-B6 herein), SEQ ID NO. 15/SEQ ID NO. 16(called VB-B10 herein), SEQ ID NO. 17/SEQ ID NO. 18 (called VB-D5herein), SEQ ID NO. 19/SEQ ID NO. 20 (called VB-D6 herein), SEQ ID NO.21/SEQ ID NO. 22 (called VB-D11 herein), SEQ ID NO. 23/SEQ ID NO. 24(called VB-E1 herein), SEQ ID NO. 25/SEQ ID NO. 26 (called VB-E2herein), SEQ ID NO. 27/SEQ ID NO. 28 (called VB-E7 herein), SEQ ID NO.29/SEQ ID NO. 30 (called VB-F2 herein), SEQ ID NO. 31/SEQ ID NO. 32(called VB-F8 herein), SEQ ID NO. 33/SEQ ID NO. 34 (called VB-G4herein), SEQ ID NO. 35/SEQ ID NO. 36 (called VB-G6 herein), SEQ ID NO.37/SEQ ID NO. 38 (called VB-H4 herein), SEQ ID NO. 39/SEQ ID NO. 40(called VB-H7 herein), SEQ ID NO. 41/SEQ ID NO. 42 (called VB-H9herein), SEQ ID NO. 43/SEQ ID NO. 44 (called RV-A9 herein), SEQ ID NO.45/SEQ ID NO. 46 (called RV-F8 herein), SEQ ID NO. 47/SEQ ID NO. 48(called RV-H2 herein), SEQ ID NO. 49/SEQ ID NO. 50 (called RV-H4herein), SEQ ID NO. 51/SEQ ID NO. 52 (called RV-H5 herein), SEQ ID NO.53/SEQ ID NO. 54 (called C1 herein), SEQ ID NO. 55/SEQ ID NO. 56 (calledVR-A2 herein), SEQ ID NO. 57/SEQ ID NO. 58 (called VR-A3 herein), SEQ IDNO. 59/SEQ ID NO. 60 (called VR-A10 herein), SEQ ID NO. 61/SEQ ID NO. 62(called VR-B2 herein), SEQ ID NO. 63/SEQ ID NO. 64 (called VR-B4herein), SEQ ID NO. 65/SEQ ID NO. 66 (called VR-B 11 herein), SEQ ID NO.67/SEQ ID NO. 68 (called VR-05 herein), SEQ ID NO. 69/SEQ ID NO. 70(called VR-C7 herein), SEQ ID NO. 71/SEQ ID NO. 72 (called VR-C11herein), SEQ ID NO. 73/SEQ ID NO. 74 (called VR-E3 herein), SEQ ID NO.75/SEQ ID NO. 76 (called VR-G11 herein), SEQ ID NO. 77/SEQ ID NO. 78(called VK-B8 herein), SEQ ID NO. 79/SEQ ID NO. 80 called VR-H9 herein),SEQ ID NO. 77/SEQ ID NO. 81 (called VK-B8A herein), and combinationsthereof.

The present disclosure provides a fully human antibody Fab fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ IDNO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71,SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO.81, and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72,SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80, andcombinations thereof. Preferably, the fully human antibody Fab fragmenthas both a heavy chain variable domain region and a light chain variabledomain region wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO.26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO.36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO.41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO.46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO.51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO.56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID NO.66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO.71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO.76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, andcombinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connecting the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO.51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ IDNO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79,SEQ ID NO. 81, and combinations thereof, and that has a light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ IDNO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60,SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO.70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ IDNO. 80, and combinations thereof. Preferably, the fully human singlechain antibody has both a heavy chain variable domain region and a lightchain variable domain region, wherein the single chain fully humanantibody has a heavy chain/light chain variable domain sequence selectedfrom the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ IDNO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ IDNO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ IDNO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ IDNO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ IDNO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ IDNO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ IDNO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ IDNO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ IDNO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ IDNO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ IDNO. 59/SEQ ID NO. 60, SEQ ID NO. 61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ IDNO. 64, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ IDNO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ IDNO. 74, SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ IDNO. 79/SEQ ID NO. 80, and combinations thereof.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers, comprising administering an effectiveamount of an anti-VEGFR2 polypeptide, wherein the anti-VEGFR2polypeptide is selected from the group consisting of a fully humanantibody of an IgG class that binds to a VEGFR2 epitope with a bindingaffinity of at least 10⁻⁶M, a fully human antibody Fab fragment, havinga variable domain region from a heavy chain and a variable domain regionfrom a light chain, a single chain human antibody, having a variabledomain region from a heavy chain and a variable domain region from alight chain and a peptide linker connecting the heavy chain and lightchain variable domain regions, and combinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ IDNO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71,SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO.81, and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72,SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80, andcombinations thereof;

wherein the fully human antibody Fab fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ IDNO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59,SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO.69, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ IDNO. 79, SEQ ID NO. 81, and combinations thereof, and that has the lightchain variable domain sequence that is at least 95% identical to theamino acid sequences selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO.22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ IDNO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50,SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO.60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ IDNO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQID NO. 80, and combinations thereof; and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO.51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ IDNO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79,SEQ ID NO. 81, and combinations thereof, and that has the light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ IDNO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60,SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO.70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ IDNO. 80, and combinations thereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO.42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO.47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO.52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO.57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO. 61/SEQ ID NO.62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO.67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO.72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID NO.77/SEQ ID NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO.81, and combinations thereof. Preferably, the fully human antibody Fabfragment has both a heavy chain variable domain region and a light chainvariable domain region wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2 (called VB-A2 herein), SEQ ID NO. 3/SEQ ID NO. 4(called VB-A3 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called VB-A7 herein),SEQ ID NO. 7/SEQ ID NO. 8 (called VBV-A7 herein), SEQ ID NO. 9/SEQ IDNO. 10 (called VB-A9 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called VB-A10herein), SEQ ID NO. 13/SEQ ID NO. 14 (called VB-B6 herein), SEQ ID NO.15/SEQ ID NO. 16 (called VB-B10 herein), SEQ ID NO. 17/SEQ ID NO. 18(called VB-D5 herein), SEQ ID NO. 19/SEQ ID NO. 20 (called VB-D6herein), SEQ ID NO. 21/SEQ ID NO. 22 (called VB-D11 herein), SEQ ID NO.23/SEQ ID NO. 24 (called VB-E1 herein), SEQ ID NO. 25/SEQ ID NO. 26(called VB-E2 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called VB-E7herein), SEQ ID NO. 29/SEQ ID NO. 30 (called VB-F2 herein), SEQ ID NO.31/SEQ ID NO. 32 (called VB-F8 herein), SEQ ID NO. 33/SEQ ID NO. 34(called VB-G4 herein), SEQ ID NO. 35/SEQ ID NO. 36 (called VB-G6herein), SEQ ID NO. 37/SEQ ID NO. 38 (called VB-H4 herein), SEQ ID NO.39/SEQ ID NO. 40 (called VB-H7 herein), SEQ ID NO. 41/SEQ ID NO. 42(called VB-H9 herein), SEQ ID NO. 43/SEQ ID NO. 44 (called RV-A9herein), SEQ ID NO. 45/SEQ ID NO. 46 (called RV-F8 herein), SEQ ID NO.47/SEQ ID NO. 48 (called RV-H2 herein), SEQ ID NO. 49/SEQ ID NO. 50(called RV-H4 herein), SEQ ID NO. 51/SEQ ID NO. 52 (called RV-H5herein), SEQ ID NO. 53/SEQ ID NO. 54 (called C1 herein), SEQ ID NO.55/SEQ ID NO. 56 (called VR-A2 herein), SEQ ID NO. 57/SEQ ID NO. 58(called VR-A3 herein), SEQ ID NO. 59/SEQ ID NO. 60 (called VR-A10herein), SEQ ID NO. 61/SEQ ID NO. 62 (called VR-B2 herein), SEQ ID NO.63/SEQ ID NO. 64 (called VR-B4 herein), SEQ ID NO. 65/SEQ ID NO. 66(called VR-B11 herein), SEQ ID NO. 67/SEQ ID NO. 68 (called VR-05herein), SEQ ID NO. 69/SEQ ID NO. 70 (called VR-C7 herein), SEQ ID NO.71/SEQ ID NO. 72 (called VR-C11 herein), SEQ ID NO. 73/SEQ ID NO. 74(called VR-E3 herein), SEQ ID NO. 75/SEQ ID NO. 76 (called VR-G11herein), SEQ ID NO. 77/SEQ ID NO. 78 (called VK-B8 herein), SEQ ID NO.79/SEQ ID NO. 80 (called VR-H9 herein), SEQ ID NO. 77/SEQ ID NO. 81(called VK-B8A herein), and combinations thereof. Preferably, the fullyhuman single chain antibody has both a heavy chain variable domainregion and a light chain variable domain region, wherein the singlechain fully human antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO.42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO.47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO.52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO.57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO. 61/SEQ ID NO.62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO.67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO.72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID NO.77/SEQ ID NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO.81, and combinations thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows SDS-PAGE analysis of VK-B8, an exemplary anti-VEGFR2antibody disclosed herein and a commercially available therapeuticmonoclonal antibody.

FIG. 2 shows how VK-B8 blocks VEGF binding to soluble VEGFR2-Fc with anIC₅₀ of about 7.7×10⁻¹⁰ M.

FIG. 3 shows VK-B8 cell binding to HUVE cells and an EC₅₀ of about1.299⁻⁹M.

FIG. 4 shows VEGF-mediated HUVEC proliferation at 100 mg/ml VEGF. VK-B8was compared to Bevacizumab (Avastin®) a marketed anti-VEGF-A (ligand)antibody. VK-B8 shows comparable efficacy to Bevacizumab (Avastin®) inthis in vitro model.

FIG. 5 shows VEGF-stimulated autophosphorylation at 100 ng/ml VEGF ofthe VEFGR2 receptor in HUVECs. Various anti-VEGFR2 antibodies werecompared at an antibody concentration of 10 μg/ml. VK-B8 is the thirdcolumn from the left.

FIG. 6 shows overlaid ANSEC (analytical size-exclusion chromatography)chromatograms of VK-B8 and Avastin® in PBS buffer at pH 6.8:STD/standard run (grey dotted line), VK-B8 spectrum (red), Avastin®spectrum (green).

FIGS. 7A and 7B show overlaid ANSEC chromatograms (Ultra Violet trace at280 nm) of VK-B8 in PBS buffer at pH 6.8 immediately after purificationand after 4 months at 4° C.: VK-B8 immediately after purificationspectrum (blue), VK-B8 after 4 months at 4° C. spectrum (green). FIG. 8Bis a zoomed in region of 8A to examine baseline fluctuations.

FIG. 8 shows an effect of two antibodies on MC38 colon tumor growth.VK-B8 was compared to Bevacizumab (Avastin®) a marketed anti-VEGF-A(ligand) antibody. VK-B8 shows superior efficacy to Bevacizumab(Avastin) in this in vivo model.

FIG. 9 shows an effect of two antibodies on A431 epidermoid carcinomacell growth. VK-B8 was compared to Bevacizumab (Avastin) a marketedanti-VEGF-A (ligand) antibody. VK-B8 shows superior efficacy toBevacizumab (Avastin®) in this in vivo model.

FIG. 10 shows the synergistic combination of an anti-VEGFR2 antibodywhen administered with an anti-EGFR antibody (Erbitux®, a humanizedanti-EGFR antibody or A6, a fully human anti-EGFR antibody) inhibitingtumor cell growth in vivo.

FIG. 11 shows the IC₅₀ value for inhibition of VEGF-induced VEGFR2activation by VK-B8. The IC₅₀ is 0.12 nM, that is, inhibition ofVEGF-mediated, VEGFR2-activating autophosphorylation.

FIG. 12 shows the IC₅₀ value for the inhibition of VEGF-induced p44/p42MAPK (Erk1/2) phosphorylation by VK-B8, or VEGF-mediated p44/p42 MAPK(Erk1/2) phosphorylation. The IC₅₀ is 0.08 nM.

FIG. 13 shows the IC₅₀ value for the inhibition of VEGF-induced HUVECcell proliferation by VK-B8. The IC₅₀ is 14.9 nM.

FIG. 14 shows the IC₅₀ value for the inhibition of VEGF-induced HUVECcell migration by VK-B8. The IC₅₀ is 0.53 nM.

FIG. 15 shows the IC₅₀ value for the inhibition of VEGF-C-induced,VEGFR2 activation by VK-B8 to show VEGF-C-mediated, VEGFR2-activatingautophosphorylation. The IC₅₀ is 1.9 nM.

DETAILED DESCRIPTION

The present disclosure provides a fully human antibody of an IgG classthat binds to a VEGFR2 epitope with a binding affinity of 100 nM orless, that has a heavy chain variable domain sequence that is at least95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ IDNO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45,SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO.55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ IDNO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 73, SEQID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46,SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO.56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ IDNO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQID NO. 76, SEQ ID NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, and combinationsthereof. Preferably, the fully human antibody has both a heavy chain anda light chain wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2 (called VB-A2 herein), SEQ ID NO. 3/SEQ ID NO. 4(called VB-A3 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called VB-A7 herein),SEQ ID NO. 7/SEQ ID NO. 8 (called VBV-A7 herein), SEQ ID NO. 9/SEQ IDNO. 10 (called VB-A9 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called VB-A10herein), SEQ ID NO. 13/SEQ ID NO. 14 (called VB-B6 herein), SEQ ID NO.15/SEQ ID NO. 16 (called VB-B10 herein), SEQ ID NO. 17/SEQ ID NO. 18(called VB-D5 herein), SEQ ID NO. 19/SEQ ID NO. 20 (called VB-D6herein), SEQ ID NO. 21/SEQ ID NO. 22 (called VB-D11 herein), SEQ ID NO.23/SEQ ID NO. 24 (called VB-E1 herein), SEQ ID NO. 25/SEQ ID NO. 26(called VB-E2 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called VB-E7herein), SEQ ID NO. 29/SEQ ID NO. 30 (called VB-F2 herein), SEQ ID NO.31/SEQ ID NO. 32 (called VB-F8 herein), SEQ ID NO. 33/SEQ ID NO. 34(called VB-G4 herein), SEQ ID NO. 35/SEQ ID NO. 36 (called VB-G6herein), SEQ ID NO. 37/SEQ ID NO. 38 (called VB-H4 herein), SEQ ID NO.39/SEQ ID NO. 40 (called VB-H7 herein), SEQ ID NO. 41/SEQ ID NO. 42(called VB-H9 herein), SEQ ID NO. 43/SEQ ID NO. 44 (called RV-A9herein), SEQ ID NO. 45/SEQ ID NO. 46 (called RV-F8 herein), SEQ ID NO.47/SEQ ID NO. 48 (called RV-H2 herein), SEQ ID NO. 49/SEQ ID NO. 50(called RV-H4 herein), SEQ ID NO. 51/SEQ ID NO. 52 (called RV-H5herein), SEQ ID NO. 53/SEQ ID NO. 54 (called C1 herein), SEQ ID NO.55/SEQ ID NO. 56 (called VR-A2 herein), SEQ ID NO. 57/SEQ ID NO. 58(called VR-A3 herein), SEQ ID NO. 59/SEQ ID NO. 60 (called VR-A10herein), SEQ ID NO. 61/SEQ ID NO. 62 (called VR-B2 herein), SEQ ID NO.63/SEQ ID NO. 64 (called VR-B4 herein), SEQ ID NO. 65/SEQ ID NO. 66(called VR-B11 herein), SEQ ID NO. 67/SEQ ID NO. 68 (called VR-05herein), SEQ ID NO. 69/SEQ ID NO. 70 (called VR-C7 herein), SEQ ID NO.71/SEQ ID NO. 72 (called VR-C11 herein), SEQ ID NO. 73/SEQ ID NO. 74(called VR-E3 herein), SEQ ID NO. 75/SEQ ID NO. 76 (called VR-G11herein), SEQ ID NO. 77/SEQ ID NO. 78 (called VK-B8 herein), SEQ ID NO.79/SEQ ID NO. 80 (called VR-H9 herein), SEQ ID NO. 77/SEQ ID NO. 81(called VK-B8 herein), and combinations thereof.

The present disclosure provides a fully human antibody Fab fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ IDNO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71,SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO.81, and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72,SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80, andcombinations thereof. Preferably, the fully human antibody Fab fragmenthas both a heavy chain variable domain region and a light chain variabledomain region wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO.26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO.36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO.41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO.46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO.51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO.56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID NO.66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO.71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO.76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, SEQ ID NO.77/SEQ ID NO. 81, and combinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO.51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ IDNO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79,SEQ ID NO. 81, and combinations thereof, and that has a light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ IDNO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60,SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO.70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ IDNO. 80, and combinations thereof. Preferably, the fully human singlechain antibody has both a heavy chain variable domain region and a lightchain variable domain region, wherein the single chain fully humanantibody has a heavy chain/light chain variable domain sequence selectedfrom the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ IDNO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ IDNO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ IDNO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ IDNO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ IDNO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ IDNO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ IDNO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ IDNO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ IDNO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ IDNO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ IDNO. 59/SEQ ID NO. 60, SEQ ID NO. 61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ IDNO. 64, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ IDNO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ IDNO. 74, SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ IDNO. 79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO. 81, and combinationsthereof.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers, comprising administering an effectiveamount of an anti-VEGFR2 polypeptide, wherein the anti-VEGFR2polypeptide is selected from the group consisting of a fully humanantibody of an IgG class that binds to a VEGFR2 epitope with a bindingaffinity of at least 10⁻⁶M, a fully human antibody Fab fragment, havinga variable domain region from a heavy chain and a variable domain regionfrom a light chain, a single chain human antibody, having a variabledomain region from a heavy chain and a variable domain region from alight chain and a peptide linker connecting the heavy chain and lightchain variable domain regions, and combinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ IDNO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71,SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO.81, and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72,SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80, andcombinations thereof;

wherein the fully human antibody Fab fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ IDNO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59,SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO.69, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ IDNO. 79, SEQ ID NO. 81, and combinations thereof, and that has the lightchain variable domain sequence that is at least 95% identical to theamino acid sequences selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO.22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ IDNO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50,SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO.60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ IDNO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQID NO. 80, and combinations thereof; and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO.51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ IDNO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79,SEQ ID NO. 81, and combinations thereof, and that has the light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ IDNO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60,SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO.70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ IDNO. 80, and combinations thereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO.42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO.47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO.52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO.57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO. 61/SEQ ID NO.62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO.67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO.72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID NO.77/SEQ ID NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO.81, and combinations thereof. Preferably, the fully human antibody Fabfragment has both a heavy chain variable domain region and a light chainvariable domain region wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ IDNO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO.26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO.36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO.41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO.46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO.51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO.56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID NO.66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO.71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO.76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, SEQ ID NO.77/SEQ ID NO. 81, and combinations thereof. Preferably, the fully humansingle chain antibody has both a heavy chain variable domain region anda light chain variable domain region, wherein the single chain fullyhuman antibody has a heavy chain/light chain variable domain sequenceselected from the group consisting SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO.3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8,SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO.13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO.18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO.23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO.28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO.33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO.38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO.43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO.48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52, SEQ ID NO.53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO.58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO. 61/SEQ ID NO. 62, SEQ ID NO.63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID NO.68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO.73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID NO.78, SEQ ID NO. 79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO. 81, andcombinations thereof.

Preferably, the mammalian cancer to be treated is selected from thegroup consisting of ovarian, colon, breast or hepatic carcinoma celllines, myelomas, neuroblastic-derived CNS tumors, monocytic leukemias,B-cell derived leukemias, T-cell derived leukemias, B-cell derivedlymphomas, T-cell derived lymphomas, mast cell derived tumors, andcombinations thereof.

By “inhibit” is meant a measurable reduction in a phenomenon, often usedherein in reference to any of the following: the interaction of VEGFwith a VEGFR, VEGF- or VEGFR-mediated angiogenesis, angiogenesis,symptoms of angiogenesis, the viability of VEGFR-containing cells, theviability of VEGF-dependent Ba/F3 cells, or VEGF- or VEGFR-mediatedcellular proliferation as compared to a control sample not treated withthe polypeptide. A polypeptide will inhibit a VEGF- or VEGFR2 mediatedactivity if the reduction in activity or interaction is at least 10%,preferably 20%, 30%, 40%, or 50%, and more preferably 60%, 70%, 80%, 90%or more.

By “VEGF biological activity” is meant any function of any VEGF familymember acting through any VEGF receptor, but particularly signalingthrough a VEGFR2 receptor. The VEGF ligand family includes VEGF-A,VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PIGF), as well asvarious alternatively spliced forms of VEGF including VEGF121, VEGF145,VEGF165, VEGF189, and VEGF206 (Tischer et al., J. Biol. Chem,266:11947-11954, 1991). The VEGFR family of tyrosine kinase receptorsincludes VEGFR-1 (also known as Flt-1), VEGFR2 (also known as KDR (humanform) or Flk-1 (mouse form)), and VEGFR-3 (also known as Flt-4). VEGFligands bind to the VEGF receptors to induce, for example, angiogenesis,vasculogenesis, endothelial cell proliferation, vasodilation, and cellmigration. VEGFR2 is believed to be the VEGFR most involved inangiogenesis. A VEGFR2 or KDR-mediated biological activity is anybiological function in which VEGFR2 or KDR participates insignificantly, such that antagonism of VEGFR2 or KDR causes a measurabledecrease in the biological activity. Methods for measuring angiogenesisare standard, and are described, for example, in Jain et al. (Nat. Rev.Cancer 2:266-276, 2002). Angiogenesis can be assayed by measuring thenumber of non-branching blood vessel segments (number of segments perunit area), the functional vascular density (total length of perfusedblood vessel per unit area), the vessel diameter, the formation ofvascular channels, or the vessel volume density (total of calculatedblood vessel volume based on length and diameter of each segment perunit area). Exemplary assays for VEGF-mediated proliferation andangiogenesis can be found in U.S. Pat. No. 6,559,126, the disclosure ofwhich is incorporated by reference herein, Lyden et al, Nature Medicine7:1194 (2001), Jacob et al, Exp. Pathol. 15:1234 (1978) and Bae et al,J. Biol. Chem. 275:13588 (2000). These assays can be performed usingeither purified receptor or ligand or both, and can be performed invitro or in vivo. These assays can also be performed in cells using agenetically introduced or the naturally-occurring ligand or receptor orboth. A polypeptide that inhibits the biological activity of VEGF willcause a decrease of at least 10%, preferably 20%, 30%, 40%, or 50%, andmore preferably 60%, 70%, 80%, 90% or greater decrease in the biologicalactivity of VEGF. The inhibition of biological activity can also bemeasured by the IC₅₀. Preferably, a polypeptide that inhibits thebiological activity of VEGF or VEGFR2 will have an IC₅₀ of less than 100nM, more preferably less than 10 nM and most preferably less than 1 nM.

Polypeptides of the present invention can be produced using any standardmethods. In one example, the polypeptides are produced by recombinantDNA methods by inserting a nucleic acid sequence (e.g., a cDNA) encodingthe polypeptide into a recombinant expression vector and expressing theDNA sequence under conditions promoting expression.

Nucleic acids encoding any of the various polypeptides disclosed hereinmay be synthesized chemically. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells. See for example: Mayfield et al., Proc.Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr.Purif. 2002 (1):96-105; Connell N D. Curr. Opin. Biotechnol. 200112(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; andSharp et al. Yeast. 1991 7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a poly-histidine tag, a FLAG tag, a myctag, an HA tag, or a GST tag. Appropriate cloning and expression vectorsfor use with bacterial, fungal, yeast, and mammalian cellular hosts canbe found in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y.,1985).

The expression construct is introduced into the host cell using a methodappropriate to the host cell. A variety of methods for introducingnucleic acids into host cells are known, including, but not limited to,electroporation; transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent). Suitable host cells include prokaryotes,yeast, mammalian cells, or bacterial cells.

Suitable bacteria include gram negative or gram positive organisms, forexample, E. coli or Bacillus spp. Yeast, preferably from theSaccharomyces species, such as S. cerevisiae, may also be used forproduction of polypeptides. Various mammalian or insect cell culturesystems can also be employed to express recombinant proteins.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, (Bio/Technology, 6:47, 1988).Examples of suitable mammalian host cell lines include endothelialcells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinesehamster ovary (CHO), human embryonic kidney cells, HeLa, 293, and BHKcell lines. Purified polypeptides are prepared by culturing suitablehost/vector systems to express the recombinant proteins. For manyapplications, the small size of many of the polypeptides disclosedherein would make expression in E. coli as the preferred method forexpression. The protein is then purified from culture media or cellextracts.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

The polypeptide can be purified by isolation/purification methods forproteins generally known in the field of protein chemistry. Non-limitingexamples include extraction, recrystallization, salting out (e.g., withammonium sulfate or sodium sulfate), centrifugation, dialysis,ultrafiltration, adsorption chromatography, ion exchange chromatography,hydrophobic chromatography, normal phase chromatography, reversed-phasechromatography, gel filtration, gel permeation chromatography, affinitychromatography, electrophoresis, countercurrent distribution or anycombinations of these. After purification, polypeptides may be exchangedinto different buffers and/or concentrated by any of a variety ofmethods known to the art, including, but not limited to, filtration anddialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

Post-Translational Modifications of Polypeptides

In certain embodiments, the binding polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modifications include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. A preferred form ofglycosylation is sialylation, which conjugates one or more sialic acidmoieties to the polypeptide. Sialic acid moieties improve solubility andserum half-life while also reducing the possible immunogeneticity of theprotein. See, e.g., Raju et al. Biochemistry. 2001 31; 40(30):8868-76.Effects of such non-amino acid elements on the functionality of apolypeptide may be tested for its antagonizing role of VEGFR2 or VEGFfunction, e.g., its inhibitory effect on angiogenesis or on tumorgrowth.

In one specific embodiment, modified forms of the subject solublepolypeptides comprise linking the subject soluble polypeptides tononproteinaceous polymers. In one specific embodiment, the polymer ispolyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes,in the manner as set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. Examples of the modifiedpolypeptide include PEGylated VK-B8.

PEG is a water soluble polymer that is commercially available or can beprepared by ring-opening polymerization of ethylene glycol according tomethods well known in the art (Sandler and Karo, Polymer Synthesis,Academic Press, New York, Vol. 3, pages 138-161). The term “PEG” is usedbroadly to encompass any polyethylene glycol molecule, without regard tosize or to modification at an end of the PEG, and can be represented bythe formula: X—O(CH₂CH₂O)_(n)-1CH₂CH₂OH (1), where n is 20 to 2300 and Xis H or a terminal modification, e.g., a C₁₋₄ alkyl. In one embodiment,the PEG of the invention terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). A PEG can contain further chemicalgroups which are necessary for binding reactions; which results from thechemical synthesis of the molecule; or which is a spacer for optimaldistance of parts of the molecule. In addition, such a PEG can consistof one or more PEG side-chains which are linked together. PEGs with morethan one PEG chain are called multiarmed or branched PEGs. Branched PEGscan be prepared, for example, by the addition of polyethylene oxide tovarious polyols, including glycerol, pentaerythriol, and sorbitol. Forexample, a four-armed branched PEG can be prepared from pentaerythrioland ethylene oxide. Branched PEG are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEGside-chains (PEG2) linked via the primary amino groups of a lysine(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).

A variety of molecular mass forms of PEG can be selected, e.g., fromabout 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), forconjugating to VEGFR2 binding polypeptides. The number of repeatingunits “n” in the PEG is approximated for the molecular mass described inDaltons. It is preferred that the combined molecular mass of PEG on anactivated linker is suitable for pharmaceutical use. Thus, in oneembodiment, the molecular mass of the PEG molecules does not exceed100,000 Da. For example, if three PEG molecules are attached to alinker, where each PEG molecule has the same molecular mass of 12,000 Da(each n is about 270), then the total molecular mass of PEG on thelinker is about 36,000 Da (total n is about 820). The molecular massesof the PEG attached to the linker can also be different, e.g., of threemolecules on a linker two PEG molecules can be 5,000 Da each (each n isabout 110) and one PEG molecule can be 12,000 Da (n is about 270).

In a specific embodiment of the invention, a VEGFR2 binding polypeptideis covalently linked to one poly(ethylene glycol) group of the formula:—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of thepoly(ethylene glycol) group forming an amide bond with one of the aminogroups of the binding polypeptide; R being lower alkyl; x being 2 or 3;m being from about 450 to about 950; and n and m being chosen so thatthe molecular weight of the conjugate minus the binding polypeptide isfrom about 10 to 40 kDa. In one embodiment, a binding polypeptide's6-amino group of a lysine is the available (free) amino group.

The above conjugates may be more specifically presented by formula (II):P—NHCO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR (II), wherein P is the group of abinding polypeptide as described herein, (i.e. without the amino groupor amino groups which form an amide linkage with the carbonyl shown informula (II); and wherein R is lower alkyl; x is 2 or 3; m is from about450 to about 950 and is chosen so that the molecular weight of theconjugate minus the binding polypeptide is from about 10 to about 40kDa. As used herein, the given ranges of “m” have an orientationalmeaning. The ranges of “m” are determined in any case, and exactly, bythe molecular weight of the PEG group.

In one specific embodiment, carbonate esters of PEG are used to form thePEG-binding polypeptide conjugates. N,N′-disuccinimidylcarbonate (DSC)may be used in the reaction with PEG to form active mixedPEG-succinimidyl carbonate that may be subsequently reacted with anucleophilic group of a linker or an amino group of a bindingpolypeptide (see U.S. Pat. Nos. 5,281,698 and 5,932,462). In a similartype of reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively.

In some embodiments, the pegylated binding polypeptide comprises a PEGmolecule covalently attached to the alpha amino group of the N-terminalamino acid. Site specific N-terminal reductive amination is described inPepinsky et al., (2001) JPET, 297, 1059, and U.S. Pat. No. 5,824,784.The use of a PEG-aldehyde for the reductive amination of a proteinutilizing other available nucleophilic amino groups is described in U.S.Pat. No. 4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254, 12579,and in Chamow et al., (1994) Bioconjugate Chem. 5, 133.

In another embodiment, pegylated binding polypeptide comprises one ormore PEG molecules covalently attached to a linker, which in turn isattached to the alpha amino group of the amino acid residue at theN-terminus of the binding polypeptide. Such an approach is disclosed inU.S. Patent Publication No. 2002/0044921 and in WO094/01451.

In one embodiment, a binding polypeptide is pegylated at the C-terminus.In a specific embodiment, a protein is pegylated at the C-terminus bythe introduction of C-terminal azido-methionine and the subsequentconjugation of a methyl-PEG-triarylphosphine compound via the Staudingerreaction. This C-terminal conjugation method is described in Cazalis etal., Bioconjug. Chem. 2004; 15(5):1005-1009.

The ratio of a binding polypeptide to activated PEG in the conjugationreaction can be from about 1:0.5 to 1:50, between from about 1:1 to1:30, or from about 1:5 to 1:15. Various aqueous buffers can be used inthe present method to catalyze the covalent addition of PEG to thebinding polypeptide. In one embodiment, the pH of a buffer used is fromabout 7.0 to 9.0. In another embodiment, the pH is in a slightly basicrange, e.g., from about 7.5 to 8.5. Buffers having a pKa close toneutral pH range may be used, e.g., phosphate buffer.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated binding polypeptide, such as size exclusion(e.g. gel filtration) and ion exchange chromatography. Products may alsobe separated using SDS-PAGE. Products that may be separated includemono-, di-, tri- poly- and un-pegylated binding polypeptide, as well asfree PEG. The percentage of mono-PEG conjugates can be controlled bypooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety percent ofmono-PEG conjugates represents a good balance of yield and activity.Compositions in which, for example, at least ninety-two percent or atleast ninety-six percent of the conjugates are mono-PEG species may bedesired. In an embodiment of this invention the percentage of mono-PEGconjugates is from ninety percent to ninety-six percent.

In one embodiment, PEGylated binding polypeptide of the inventioncontain one, two or more PEG moieties. In one embodiment, the PEGmoiety(ies) are bound to an amino acid residue which is on the surfaceof the protein and/or away from the surface that contacts the targetligand. In one embodiment, the combined or total molecular mass of PEGin PEG-binding polypeptide is from about 3,000 Da to 60,000 Da,optionally from about 10,000 Da to 36,000 Da. In a one embodiment, thePEG in pegylated binding polypeptide is a substantially linear,straight-chain PEG.

In one embodiment of the invention, the PEG in pegylated bindingpolypeptide is not hydrolyzed from the pegylated amino acid residueusing a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5) over 8to 16 hours at room temperature, and is thus stable. In one embodiment,greater than 80% of the composition is stable mono-PEG-bindingpolypeptide, more preferably at least 90%, and most preferably at least95%.

In another embodiment, the pegylated binding polypeptides of theinvention will preferably retain at least 25%, 50%, 60%, 70% least 80%,85%, 90%, 95% or 100% of the biological activity associated with theunmodified protein. In one embodiment, biological activity refers to itsability to bind to VEGFR2, as assessed by KD, k_(on), or k_(off). In onespecific embodiment, the pegylated binding polypeptide protein shows anincrease in binding to VEGFR relative to unpegylated bindingpolypeptide.

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t.sub.1/2) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf-life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

Therapeutic Formulations and Modes of Administration

The present disclosure features methods for treating conditions orpreventing pre-conditions which respond to an inhibition of VEGFbiological activity. Preferred examples are conditions that arecharacterized by inappropriate angiogenesis. Techniques and dosages foradministration vary depending on the type of specific polypeptide andthe specific condition being treated but can be readily determined bythe skilled artisan. In general, regulatory agencies require that aprotein reagent to be used as a therapeutic is formulated so as to haveacceptably low levels of pyrogens. Accordingly, therapeutic formulationswill generally be distinguished from other formulations in that they aresubstantially pyrogen free, or at least contain no more than acceptablelevels of pyrogen as determined by the appropriate regulatory agency(e.g., FDA).

Therapeutic compositions of the present disclosure may be administeredwith a pharmaceutically acceptable diluent, carrier, or excipient, inunit dosage form. Administration may be parenteral (e.g., intravenous,subcutaneous), oral, or topical, as non-limiting examples. In addition,any gene therapy technique, using nucleic acids encoding thepolypeptides of the invention, may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; or a liquid forintravenous, subcutaneous or parenteral administration; gel, lotion,ointment, cream, or a polymer or other sustained release vehicle forlocal administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the polypeptideis administered at about 0.01 μg/kg to about 50 mg/kg per day,preferably 0.01 mg/kg to about 30 mg/kg per day, and most preferably 0.1mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or preferably lessfrequently (e.g., weekly, every two weeks, every three weeks, monthly,or quarterly). In addition, as is known in the art, adjustments for ageas well as the body weight, general health, sex, diet, time ofadministration, drug interaction, and the severity of the disease may benecessary.

Exemplary Uses

The VEGFR2 binding proteins described herein and their related variantsare useful in a number of therapeutic and diagnostic applications. Theseinclude the inhibition of the biological activity of VEGF by competingfor or blocking the binding to a VEGFR2.

On the basis of their efficacy as inhibitors of VEGF biologicalactivity, the polypeptides of the invention are effective against anumber of conditions associated with inappropriate angiogenesis,including but not limited to autoimmune disorders (e.g., rheumatoidarthritis, inflammatory bowel disease or psoriasis); cardiac disorders(e.g., atherosclerosis or blood vessel restenosis); retinopathies (e.g.,proliferative retinopathies generally, diabetic retinopathy, age-relatedmacular degeneration or neovascular glaucoma), renal disease (e.g.,diabetic nephropathy, malignant nephrosclerosis, thromboticmicroangiopathy syndromes; transplant rejection; inflammatory renaldisease; glomerulonephritis; mesangioproliferative glomerulonephritis;haemolytic-uraemic syndrome; and hypertensive nephrosclerosis);hemangioblastoma; hemangiomas; thyroid hyperplasias; tissuetransplantations; chronic inflammation; Meigs's syndrome; pericardialeffusion; pleural effusion; autoimmune diseases; diabetes;endometriosis; chronic asthma; undesirable fibrosis (particularlyhepatic fibrosis) and cancer, as well as complications arising fromcancer, such as pleural effusion and ascites. Preferably, theVEGFR2-binding polypeptides of the invention can be used for thetreatment or prevention of hyperproliferative diseases or cancer and themetastatic spread of cancers. Non-limiting examples of cancers includebladder, blood, bone, brain, breast, cartilage, colon kidney, liver,lung, lymph node, nervous tissue, ovary, pancreatic, prostate, skeletalmuscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid,trachea, urogenital tract, ureter, urethra, uterus, or vaginal cancer.Additional treatable conditions can be found in U.S. Pat. No. 6,524,583,incorporated by reference herein. Other references describing uses forVEGFR2 binding polypeptides include: McLeod et al., Invest. Ophthalmol.Vis. Sci. 2002; 43(2):474-82; Watanabe et al., Exp. Dermatol. 2004;13(11):671-81; Yoshiji et al., Gut. 2003 52(9):1347-54; Verheul et al.,Oncologist. 2000; 5 Suppl 1:45-50; and Boldicke et al., Stem Cells. 200119(1):24-36.

As described herein, angiogenesis-associated diseases include, but arenot limited to, angiogenesis-dependent cancer, including, for example,solid tumors, blood born tumors such as leukemias, and tumor metastases;benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; inflammatorydisorders such as immune and non-immune inflammation; chronic articularrheumatism and psoriasis; ocular angiogenic diseases, for example,diabetic retinopathy, retinopathy of prematurity, macular degeneration,corneal graft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation and wound healing; telangiectasia psoriasisscleroderma, pyogenic granuloma, coronary collaterals, ischemic limbangiogenesis, corneal diseases, rubeosis, arthritis, diabeticneovascularization, fractures, vasculogenesis, hematopoiesis.

A VEGFR2 binding polypeptide can be administered alone or in combinationwith one or more additional therapies such as chemotherapy,radiotherapy, immunotherapy, surgical intervention, or any combinationof these. Long-term therapy is equally possible as is adjuvant therapyin the context of other treatment strategies, as described above.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject anti-VEGFR2 antibodies agents of theinvention can be used alone. Alternatively, the subject agents may beused in combination with other conventional anti-cancer therapeuticapproaches directed to treatment or prevention of proliferativedisorders (e.g., tumor). For example, such methods can be used inprophylactic cancer prevention, prevention of cancer recurrence andmetastases after surgery, and as an adjuvant of other conventionalcancer therapy. The present disclosure recognizes that the effectivenessof conventional cancer therapies (e.g., chemotherapy, radiation therapy,phototherapy, immunotherapy, and surgery) can be enhanced through theuse of a subject polypeptide therapeutic agent.

A wide array of conventional compounds has been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a polypeptide therapeutic agent of the present invention isadministered in combination with another conventional anti-neoplasticagent, either concomitantly or sequentially, such therapeutic agent maybe found to enhance the therapeutic effect of the anti-neoplastic agentor overcome cellular resistance to such anti-neoplastic agent. Thisallows decrease of dosage of an anti-neoplastic agent, thereby reducingthe undesirable side effects, or restores the effectiveness of ananti-neoplastic agent in resistant cells.

Pharmaceutical compounds that may be used for combinatory anti-tumortherapy include, merely to illustrate: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

Certain chemotherapeutic anti-tumor compounds may be categorized bytheir mechanism of action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (e.g., VEGF inhibitors, fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylpednisolone, prednisone, and prenisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disruptors.

In certain embodiments, pharmaceutical compounds that may be used forcombinatory anti-angiogenesis therapy include: (1) inhibitors of releaseof “angiogenic molecules,” such as bFGF (basic fibroblast growthfactor); (2) neutralizers of angiogenic molecules, such as an anti-βFGFantibodies; and (3) inhibitors of endothelial cell response toangiogenic stimuli, including collagenase inhibitor, basement membraneturnover inhibitors, angiostatic steroids, fungal-derived angiogenesisinhibitors, platelet factor 4, thrombospondin, arthritis drugs such asD-penicillamine and gold thiomalate, vitamin D₃ analogs,alpha-interferon, and the like. For additional proposed inhibitors ofangiogenesis, see Blood et al., Bioch. Biophys. Acta. 1032:89-118(1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab.Invest., 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885; 5,112,946;5,192,744; 5,202,352; and 6,573,256. In addition, there are a widevariety of compounds that can be used to inhibit angiogenesis, forexample, endostatin protein or derivatives, lysine binding fragments ofangiostatin, melanin or melanin-promoting compounds, plasminogenfragments (e.g., Kringles 1-3 of plasminogen), tropoin subunits,antagonists of vitronectin α_(v)β, peptides derived from Saposin B,antibiotics or analogs (e.g., tetracycline, or neomycin),dienogest-containing compositions, compounds comprising a MetAP-2inhibitory core coupled to a peptide, the compound EM-138, chalcone andits analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos.6,395,718; 6,462,075; 6,465,431; 6,475,784; 6,482,802; 6,482,810;6,500,431; 6,500,924; 6,518,298; 6,521,439; 6,525,019; 6,538,103;6,544,758; 6,544,947; 6,548,477; 6,559,126; and 6,569,845).

Depending on the nature of the combinatory therapy, administration ofthe polypeptide therapeutic agents may be continued while the othertherapy is being administered and/or thereafter. Administration of thepolypeptide therapeutic agents may be made in a single dose, or inmultiple doses. In some instances, administration of the polypeptidetherapeutic agents is commenced at least several days prior to theconventional therapy, while in other instances, administration is beguneither immediately before or at the time of the administration of theconventional therapy.

The VEGFR2 binding proteins described herein can also be detectablylabeled and used to contact cells expressing VEGFR2 for imagingapplications or diagnostic applications. For diagnostic purposes, thepolypeptide of the invention is preferably immobilized on a solidsupport. Preferred solid supports include columns (for example, affinitycolumns, such as agarose-based affinity columns), microchips, or beads.

In one example of a diagnostic application, a biological sample, such asserum or a tissue biopsy, from a patient suspected of having a conditioncharacterized by inappropriate angiogenesis is contacted with adetectably labeled polypeptide of the invention to detect levels ofVEGFR2. The levels of VEGFR2 detected are then compared to levels ofVEGFR2 detected in a normal sample also contacted with the labeledpolypeptide. An increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% in the levels of the VEGFR2 may be considered a diagnosticindicator of a condition characterized by inappropriate angiogenesis.

In certain embodiments, the VEGFR2 binding polypeptides of the inventionare further attached to a label that is able to be detected (e.g., thelabel can be a radioisotope, fluorescent compound, enzyme or enzymeco-factor). The active moiety may be a radioactive agent, such as:radioactive heavy metals such as iron chelates, radioactive chelates ofgadolinium or manganese, positron emitters of oxygen, nitrogen, iron,carbon, or gallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ¹²³I, ¹²⁵I, ¹³¹I,¹³²I, or ⁹⁹Tc. A binding agent affixed to such a moiety may be used asan imaging agent and is administered in an amount effective fordiagnostic use in a mammal such as a human and the localization andaccumulation of the imaging agent is then detected. The localization andaccumulation of the imaging agent may be detected by radioscintigraphy,nuclear magnetic resonance imaging, computed tomography or positronemission tomography. Immunoscintigraphy using VEGFR2 bindingpolypeptides directed at VEGFR2 may be used to detect and/or diagnosecancers and vasculature. For example, any of the binding polypeptideagainst the VEGFR2 marker labeled with ⁹⁹Technetium, ¹¹¹Indium, or¹²⁵Iodine may be effectively used for such imaging. As will be evidentto the skilled artisan, the amount of radioisotope to be administered isdependent upon the radioisotope. Those having ordinary skill in the artcan readily formulate the amount of the imaging agent to be administeredbased upon the specific activity and energy of a given radionuclide usedas the active moiety. Typically a person skilled in the art administers0.1-100 millicuries per dose of imaging agent, preferably 1-10millicuries, most often 2-5 millicuries. Thus, compositions according tothe present invention useful as imaging agents comprising a targetingmoiety conjugated to a radioactive moiety comprise 0.1-100 millicuries,in some embodiments preferably 1-10 millicuries, in some embodimentspreferably 2-5 millicuries, in some embodiments more preferably 1-5millicuries.

The VEGFR2 binding polypeptides can also be used to deliver additionaltherapeutic agents (including but not limited to drug compounds,chemotherapeutic compounds, and radiotherapeutic compounds) to a cell ortissue expressing VEGFR2. In one example, the VEGFR2 binding polypeptideis fused to a chemotherapeutic agent for targeted delivery of thechemotherapeutic agent to a tumor cell or tissue expressing VEGFR2.

The VEGFR2 binding polypeptides are useful in a variety of applications,including research, diagnostic and therapeutic applications. Forinstance, they can be used to isolate and/or purify receptor or portionsthereof, and to study receptor structure (e.g., conformation) andfunction.

In certain aspects, the various binding polypeptides can be used todetect or measure the expression of VEGFR2, for example, on endothelialcells (e.g., venous endothelial cells), or on cells transfected with aVEGFR2 gene. Thus, they also have utility in applications such as cellsorting and imaging (e.g., flow cytometry, and fluorescence activatedcell sorting), for diagnostic or research purposes.

In certain embodiments, the binding polypeptides of fragments thereofcan be labeled or unlabeled for diagnostic purposes. Typically,diagnostic assays entail detecting the formation of a complex resultingfrom the binding of a binding polypeptide to VEGFR2. The bindingpolypeptides or fragments can be directly labeled, similar toantibodies. A variety of labels can be employed, including, but notlimited to, radionuclides, fluorescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and4,098,876). When unlabeled, the binding polypeptides can be used inassays, such as agglutination assays. Unlabeled binding polypeptides canalso be used in combination with another (one or more) suitable reagentwhich can be used to detect the binding polypeptide, such as a labeledantibody reactive with the binding polypeptide or other suitable reagent(e.g., labeled protein A).

In one embodiment, the binding polypeptides of the present invention canbe utilized in enzyme immunoassays, wherein the subject polypeptides areconjugated to an enzyme. When a biological sample comprising a VEGFR2protein is combined with the subject binding polypeptides, bindingoccurs between the binding polypeptides and the VEGFR2 protein. In oneembodiment, a sample containing cells expressing a VEGFR2 protein (e.g.,endothelial cells) is combined with the subject antibodies, and bindingoccurs between the binding polypeptides and cells bearing a VEGFR2protein recognized by the binding polypeptide. These bound cells can beseparated from unbound reagents and the presence of the bindingpolypeptide-enzyme conjugate specifically bound to the cells can bedetermined, for example, by contacting the sample with a substrate ofthe enzyme which produces a color or other detectable change when actedon by the enzyme. In another embodiment, the subject bindingpolypeptides can be unlabeled, and a second, labeled polypeptide (e.g.,an antibody) can be added which recognizes the subject bindingpolypeptide.

In certain aspects, kits for use in detecting the presence of a VEGFR2protein in a biological sample can also be prepared. Such kits willinclude a VEGFR2 binding polypeptide which binds to a VEGFR2 protein orportion of said receptor, as well as one or more ancillary reagentssuitable for detecting the presence of a complex between the bindingpolypeptide and the receptor protein or portions thereof. Thepolypeptide compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The binding polypeptides and/orantibodies, which can be labeled or unlabeled, can be included in thekits with adjunct ingredients (e.g., buffers, such as Tris, phosphateand carbonate, stabilizers, excipients, biocides and/or inert proteins,e.g., bovine serum albumin). For example, the binding polypeptidesand/or antibodies can be provided as a lyophilized mixture with theadjunct ingredients, or the adjunct ingredients can be separatelyprovided for combination by the user. Generally these adjunct materialswill be present in less than about 5% weight based on the amount ofactive binding polypeptide or antibody, and usually will be present in atotal amount of at least about 0.001% weight based on polypeptide orantibody concentration. Where a second antibody capable of binding tothe binding polypeptide is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

Similarly, the present disclosure also provides a method of detectingand/or quantitating expression of VEGFR2, wherein a compositioncomprising a cell or fraction thereof (e.g., membrane fraction) iscontacted with a binding polypeptide which binds to a VEGFR2 or portionof the receptor under conditions appropriate for binding thereto, andthe binding is monitored. Detection of the binding polypeptide,indicative of the formation of a complex between binding polypeptide andVEGFR2 or a portion thereof, indicates the presence of the receptor.Binding of a polypeptide to the cell can be determined by standardmethods, such as those described in the working examples. The method canbe used to detect expression of VEGFR2 on cells from an individual.Optionally, a quantitative expression of VEGFR2 on the surface ofendothelial cells can be evaluated, for instance, by flow cytometry, andthe staining intensity can be correlated with disease susceptibility,progression or risk.

The present disclosure also provides a method of detecting thesusceptibility of a mammal to certain diseases. To illustrate, themethod can be used to detect the susceptibility of a mammal to diseaseswhich progress based on the amount of VEGFR2 present on cells and/or thenumber of VEGFR2-positive cells in a mammal. In one embodiment, theinvention relates to a method of detecting susceptibility of a mammal toa tumor. In this embodiment, a sample to be tested is contacted with abinding polypeptide which binds to a VEGFR2 or portion thereof underconditions appropriate for binding thereto, wherein the sample comprisescells which express VEGFR2 in normal individuals. The binding and/oramount of binding is detected, which indicates the susceptibility of theindividual to a tumor, wherein higher levels of receptor correlate withincreased susceptibility of the individual to a tumor.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The terms “VEGFR2 inhibitor” and “VEGFR2 antagonist” are usedinterchangeably. Each is a molecule that detectably inhibits at leastone function of VEGFR2. Conversely, a “VEGFR2 agonist” is a moleculethat detectably increases at least one function of VEGFR2. Theinhibition caused by a VEGFR2 inhibitor need not be complete so long asit is detectable using an assay. Any assay of a function of VEGFR2 canbe used, examples of which are provided herein. Examples of functions ofVEGFR2 that can be inhibited by a VEGFR2 inhibitor, or increased by aVEGFR2 agonist, include cancer cell growth or apoptosis (programmed celldeath), and so on. Examples of types of VEGFR2 inhibitors and VEGFR2agonists include, but are not limited to, VEGFR2 binding polypeptidessuch as antigen binding proteins (e.g., VEGFR2 inhibiting antigenbinding proteins), antibodies, antibody fragments, and antibodyderivatives.

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

A “variant” of a polypeptide (for example, an antibody) comprises anamino acid sequence wherein one or more amino acid residues are insertedinto, deleted from and/or substituted into the amino acid sequencerelative to another polypeptide sequence. Disclosed variants include,for example, fusion proteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and glycosylation. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Preferably, the anti-VEGFR2 antibodies disclosed herein arecharacterized by their variable domain region sequences in the heavyV_(H) and light V_(L) amino acid sequences. The preferred antibody isVK-B8, which is a kappa IgG antibody. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. See generally, Fundamental Immunology Ch. 7(Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). The variable regionsof each light/heavy chain pair form the antibody binding site such thatan intact immunoglobulin has two binding sites.

The variable regions of naturally occurring immunoglobulin chainsexhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarity determining regions or CDRs. From N-terminus toC-terminus, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat et al. inSequences of Proteins of Immunological Interest, 5.sup.th Ed., US Dept.of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242,1991. Other numbering systems for the amino acids in immunoglobulinchains include IMGT® (International ImMunoGeneTics information system;Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honeggerand Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion (Fab) thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),and complementarity determining region (CDR) fragments, single-chainantibodies (scFv), chimeric antibodies, diabodies, triabodies,tetrabodies, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H1) domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634 and 6,696,245, thedisclosures of which are incorporated by reference herein).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (Bird et al., 1988, Science242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-83).

Diabodies are bivalent antibodies comprising two polypeptide chains,wherein each polypeptide chain comprises V_(H) and V_(L) domains joinedby a linker that is too short to allow for pairing between two domainson the same chain, thus allowing each domain to pair with acomplementary domain on another polypeptide chain (Holliger et al.,1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994,Structure 2:1121-23). If the two polypeptide chains of a diabody areidentical, then a diabody resulting from their pairing will have twoidentical antigen binding sites. Polypeptide chains having differentsequences can be used to make a diabody with two different antigenbinding sites. Similarly, tribodies and tetrabodies are antibodiescomprising three and four polypeptide chains, respectively, and formingthree and four antigen binding sites, respectively, which can be thesame or different.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified (Sequences of Proteins ofImmunological Interest, 5.sup.th Ed., US Dept. of Health and HumanServices, PHS, NIH, NIH Publication no. 91-3242, 1991). Other numberingsystems for the amino acids in immunoglobulin chains include IMGT®(International ImMunoGeneTics information system; Lefranc et al., Dev.Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J.Mol. Biol. 309(3):657-670; 2001). One or more CDRs may be incorporatedinto a molecule either covalently or noncovalently to make it an antigenbinding protein. An antigen binding protein may incorporate the CDR(s)as part of a larger polypeptide chain, may covalently link the CDR(s) toanother polypeptide chain, or may incorporate the CDR(s) noncovalently.The CDRs permit the antigen binding protein to specifically bind to aparticular antigen of interest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have each andevery variable and constant regions derived from human immunoglobulinsequences. In one embodiment, fully human antibody, all of the variableand constant domains are derived from human immunoglobulin sequences (afully human antibody).

A humanized antibody has a sequence that differs from the sequence of anantibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297;5,886,152; and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies.

Further, the framework regions may be derived from one of the sameanti-VEGFR2 antibodies, from one or more different antibodies, such as ahuman antibody, or from a humanized antibody. In one example of achimeric antibody, a portion of the heavy and/or light chain isidentical with, homologous to, or derived from an antibody from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with,homologous to, or derived from an antibody (-ies) from another speciesor belonging to another antibody class or subclass. Also included arefragments of such antibodies that exhibit the desired biologicalactivity (U.S. Pat. No. 4,816,567)

A “neutralizing antibody” or an “inhibitory antibody” is an antibodythat inhibits the activation of VEGFR2 when an excess of the anti-VEGFR2antibody reduces the amount of activation or inhibition by at leastabout 20% using an assay such as those described herein in the Examples.In various embodiments, the antigen binding protein reduces the amountof amount of activation of VEGFR2 by at least 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques well-known in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function.

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human VEGFR2) if it binds to the antigen with a dissociation constant of1 nM or less.

An “antigen binding domain, “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent homology” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

A “host cell” is a cell that can be used to express a nucleic acid. Ahost cell can be a prokaryote, for example, E. coli, or it can be aeukaryote, for example, a single-celled eukaryote (e.g., a yeast orother fungus), a plant cell (e.g., a tobacco or tomato plant cell), ananimal cell (e.g., a human cell, a monkey cell, a hamster cell, a ratcell, a mouse cell, or an insect cell) or a hybridoma. Examples of hostcells include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivativessuch as Veggie CHO and related cell lines which grow in serum-free media(Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B11,which is deficient in DHFR (Urlaub et al., 1980, Proc. Natl. Acad. Sci.USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNAcell line derived from the African green monkey kidney cell line CV1(ATCC CCL 70) (McMahan et al., 1991, EMBO J. 10:2821), human embryonickidney cells such as 293,293 EBNA or MSR 293, human epidermal A431cells, human Colo205 cells, other transformed primate cell lines, normaldiploid cells, cell strains derived from in vitro culture of primarytissue, primary explants, HL-60, U937, HaK or Jurkat cells. Typically, ahost cell is a cultured cell that can be transformed or transfected witha polypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Antigen Binding Proteins

Antigen binding proteins (e.g., antibodies, antibody fragments, antibodyderivatives, antibody muteins, and antibody variants) are polypeptidesthat bind to VEGFR2, (preferably, human VEGFR2). Antigen bindingproteins include antigen binding proteins that inhibit a biologicalactivity of VEGFR2.

Oligomers that contain one or more antigen binding proteins may beemployed as VEGFR2 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding proteins arecontemplated for use, with one example being a homodimer. Otheroligomers include

One embodiment is directed to a dimer comprising two fusion proteinscreated by fusing a VEGFR2 binding fragment of an anti-VEGFR2 antibodyto the Fc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yield thedimer.

The term “Fc polypeptide” includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization also are included. Fusion proteins comprising Fc moieties(and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al., 1994, FEBS Letters 344:191. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al., 1994,Semin. Immunol. 6:267-78. In one approach, recombinant fusion proteinscomprising an anti-VEGFR2 antibody fragment or derivative fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomeric anti-VEGFR2 antibody fragments or derivatives thatform are recovered from the culture supernatant.

The present disclosure provides a VEGFR2 antigen binding protein (forexample, an anti-VEGFR2 antibody), that has one or more of the followingcharacteristics: binds to both human and murine VEGFR2, inhibits theactivation of human VEGFR2, inhibits the activation of murine VEGFR2,and binds to or near the ligand binding domain of VEGFR2.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)₂ fragments.

The present disclosure provides monoclonal antibodies that bind toVEGFR2. Monoclonal antibodies may be produced using any technique knownin the art, e.g., by immortalizing spleen cells harvested from thetransgenic animal after completion of the immunization schedule. Thespleen cells can be immortalized using any technique known in the art,e.g., by fusing them with myeloma cells to produce hybridomas. Myelomacells for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Examples of suitable cell lines for use in mouse fusionsinclude Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; examples of celllines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and48210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6.

Antigen binding proteins directed against VEGFR2 can be used, forexample, in assays to detect the presence of VEGFR2 polypeptides, eitherin vitro or in vivo. The antigen binding proteins also may be employedin purifying VEGFR2 proteins by immunoaffinity chromatography. Thoseantigen binding proteins that additionally can block ligandbinding-mediated activation of VEGFR2 may be used to inhibit abiological activity that results from such binding. Blocking antigenbinding proteins can be used in the methods disclosed herein. Suchantigen binding proteins that function as VEGFR2 antagonists may beemployed in treating any VEGFR2-induced condition, including but notlimited to various cancers.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit a VEGFR2-induced biological activity.Disorders caused or exacerbated (directly or indirectly) by theactivation of VEGFR2, examples of which are provided herein, thus may betreated. In one embodiment, the present invention provides a therapeuticmethod comprising in vivo administration of a VEGFR2 blocking antigenbinding protein to a mammal in need thereof in an amount effective forreducing a VEGFR2-induced biological activity.

Antigen binding proteins include fully human monoclonal antibodies thatinhibit a biological activity of VEGFR2, such as angiogenesis.

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, they may be purified from cellsthat naturally express them (e.g., an antibody can be purified from ahybridoma that produces it), or produced in recombinant expressionsystems, using any technique known in the art. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) ofVEGFR2 bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-VEGFR2 antibodypolypeptides substantially free of contaminating endogenous materials.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain techniquesinvolve isolating a nucleic acid encoding a polypeptide chain (orportion thereof) of an antigen binding protein of interest (e.g., ananti-VEGFR2 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype (Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover,if an IgG4 is desired, it may also be desired to introduce a pointmutation (CPSCP->CPPCP) in the hinge region (Bloom et al., 1997, ProteinScience 6:407) to alleviate a tendency to form intra-H chain disulfidebonds that can lead to heterogeneity in the IgG4 antibodies.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for VEGFR2 of at least 10⁶ nM.In other embodiments, the antigen binding proteins exhibit a K_(a) of atleast 10⁷, at least 10⁸, at least 10⁹, or at least 10¹⁰ M. In anotherembodiment, the antigen binding protein exhibits a K_(a) substantiallythe same as that of an antibody described herein in the Examples.

In another embodiment, the present disclosure provides an antigenbinding protein that has a low dissociation rate from VEGFR2. In oneembodiment, the antigen binding protein has a K_(off) of 1×10⁻⁴ to1×10⁻¹M or lower. In another embodiment, the K_(off) is 5×10⁻⁵ to5×10⁻¹M or lower. In another embodiment, the K_(off) is substantiallythe same as an antibody described herein in the Examples. In anotherembodiment, the antigen binding protein binds to VEGFR2 withsubstantially the same K_(off) as an antibody described herein in theExamples.

In another aspect, the present disclosure provides a VEGFR2 membranebinding protein. In one embodiment, the antigen binding protein has anIC₅₀ of 1000 nM or lower. In another embodiment, the IC₅₀ is 100 nM orlower; in another embodiment, the IC₅₀ is 10 nM or lower. In anotherembodiment, the IC₅₀ is substantially the same as that of an antibodydescribed herein in the Examples. In another embodiment, the antigenbinding protein inhibits an activity of VEGFR2 with substantially thesame IC₅₀ as an antibody described herein in the Examples.

In another aspect, the present disclosure provides an antigen bindingprotein that binds to human VEGFR2 expressed on the surface of a celland, when so bound, inhibits VEGFR2 signaling activity in the cell. Anymethod for determining or estimating the amount of VEGFR2 on the surfaceand/or in the interior of the cell can be used. In other embodiments,binding of the antigen binding protein to the VEGFR2-expressing cellcauses less than about 75%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, or0.1% of the cell-surface VEGFR2 to be internalized.

In another aspect, the present disclosure provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO00/09560, incorporated by reference herein.

The present disclosure further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that binds to two different epitopes of VEGFR2, or to anepitope of VEGFR2 and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise a VEGFR2 binding site from oneof the herein-described antibodies and a second VEGFR2 binding regionfrom another of the herein-described antibodies, including thosedescribed herein by reference to other publications. Alternatively, abispecific antigen binding protein may comprise an antigen binding sitefrom one of the herein described antibodies and a second antigen bindingsite from another VEGFR2 antibody that is known in the art, or from anantibody that is prepared by known methods or the methods describedherein.

Numerous methods of preparing bispecific antibodies are known in theart. Such methods include the use of hybrid-hybridomas as described byMilstein et al., 1983, Nature 305:537, and chemical coupling of antibodyfragments (Brennan et al., 1985, Science 229:81; Glennie et al., 1987,J. Immunol. 139:2367; U.S. Pat. No. 6,010,902). Moreover, bispecificantibodies can be produced via recombinant means, for example by usingleucine zipper moieties (i.e., from the Fos and Jun proteins, whichpreferentially form heterodimers; Kostelny et al., 1992, J. Immunol.148:1547) or other lock and key interactive domain structures asdescribed in U.S. Pat. No. 5,582,996. Additional useful techniquesinclude those described in U.S. Pat. Nos. 5,959,083; and 5,807,706.

In another aspect, the antigen binding protein comprises a derivative ofan antibody. The derivatized antibody can comprise any molecule orsubstance that imparts a desired property to the antibody, such asincreased half-life in a particular use. The derivatized antibody cancomprise, for example, a detectable (or labeling) moiety (e.g., aradioactive, colorimetric, antigenic or enzymatic molecule), adetectable bead (such as a magnetic or electrodense (e.g., gold bead), amolecule that binds to another molecule (e.g., biotin or streptavidin),a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, orpharmaceutically active moiety), or a molecule that increases thesuitability of the antibody for a particular use (e.g., administrationto a subject, such as a human subject, or other in vivo or in vitrouses). Examples of molecules that can be used to derivatize an antibodyinclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antibodies can beprepared using techniques well known in the art. In one embodiment, theantibody is conjugated or otherwise linked to transthyretin (TTR) or aTTR variant. The TTR or TTR variant can be chemically modified with, forexample, a chemical selected from the group consisting of dextran,poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols and polyvinyl alcohols.

Example 1

This example illustrates production runs and how to make anti-VEGFR2antibody VK-B8. The expression cassette coding for the heavy chain anddihydrofolate reductase (DHFR) was driven by the CMV major immediateearly promoter. The expression cassette coding for the light chain andneomycin phosphotransferase was driven by the CMV major immediate earlypromoter. Transfected cells were cultured in CD OptiCHO medium (LifeTechnologies) without nucleosides prior to Fluorescence-Activated CellSorting (FACS). Cells were “sorted” once using FACS to enrich for highproducers. After FACS enrichment, the cell pool was cultured andpassaged until the cell viability was greater than 90%. For productionof VK-B8, cells were scaled to a desirable density and volume at 37° C.and then placed at 28° C. for the duration of the production run.

VK-B8 was produced in Chinese hamster ovary (CHO) cells stablytransfected with expression vectors containing the human VK-B8 mAb IgG1heavy and kappa light chain structural genes. The host cell line usedfor the construction of the cell line was a Chinese hamster ovarydihydrofolate reductase (dhfr)-deficient host cell line, CHO-DG44 (LifeTechnologies). The cell line was maintained in shake flasks androutinely passaged every 3 to 4 days using DG44 (Life Technologies)medium and supplemented with PLURONIC F-68 (Life Technologies) andGlutaMAX I-CTS (Life Technologies). Two plasmids were used to generatethe stable pool; the heavy chain and light chain expression vectorspIRES.VEGFR2.VKB8.HC and pIRES.VEGFR2.VKB8.LC, respectively. The plasmidcoding for the heavy chain also coded for dhfr as a second cistron whichwas used as a selectable and amplifiable marker. The plasmid coding forthe light chain also contains neomycin phosphotransferase as a secondcistron which was used as a selectable marker. The vectors wereco-transfected into the host cell line using the cationic lipidFreestyle Max and selected for DHFR and neomycin phosphotransferaseexpression.

Example 2

This example illustrates a differential scanning calorimetry studymeasuring thermodynamic stability of the anti-VEGFR2 monoclonal antibodyVK-B8, compared with Bevacizumab (Avastin®), using MicroCal™ DSC (GEHealthcare). A thermogram for each antibody (0.48-0.55 mg/mL) wasobtained in PBS buffer, pH 7.4 from 25° C. to 85° C., using a scan rateof 1° C. per minute, unless otherwise mentioned. The VK-B8 meltingtemperature profile was monitored in PBS buffer at both pH 7.4 and 6.

IgG Melting Temperature in ° C. Avastin 71.23 VK-B8 74.78

An Analytical Size-Exclusion Chromatography (ANSEC) (Water'sBreeze-HPLC) analysis of the monoclonal antibodies VK-B8 and Bevacizumab(Avastin®) was performed in PBS buffer, pH 6.8 at 0.5 mL/min flow rateusing TSKgel Supper SW3000 column (TOSOH Biosciences). Aggregateformation of VK-B8 in PBS buffer at 4° C. was monitored for 4 monthsusing the same system. IgG samples were compared with the BIO-RAD gelfiltration protein standard (Cat #, 151-1901: Thyroglobi=670 KDa;Gamma-globulin=158 KDa; Ovalbumin=44 KDa; Myoglobin=17 KDa; VitaminB12=1.35 KDa). The results are shown in FIG. 7, which provide overlaidANSEC chromatograms (Ultra Violet trace at 280 nm) of VK-B8 andBevacizumab (Avastin) in PBS buffer at pH 6.8; STD/standard run (greydotted line), VK-B8 spectrum (red), Bevacizumab (Avastin) spectrum(green).

ANSEC analysis of the VK-B8 IgG sample at 4° C. after 0 days and 4months is shown in FIGS. 7A and 7B which show overlaid ANSEC analysis(Ultra Violet trace at 280 nm0 days (blue) and 4 months (green)) A:representative full spectra, B: Enlarged version of A indicating thatVK-B8 remained intact after 4 months.

Further, a Biacore analysis of four antibodies was done for binding.

Biacore analysis of several VEGFR-2 antibodies, including VK-B8

mAb ka (1/Ms) kd (1/s) Kd (M) RV-H5 2.76E+06 2.25E−03  8.15E−10 VB-A33.88E+06 1.20E−02  3.09E−09 VB-A9 1.64E+05 4.96E−04  3.02E−09 VK-B87.24E+05 8.73E−05 1.21.E−10

Example 3

This example illustrates in vivo data comparing a marketed anti-VEGF(ligand) humanized monoclonal antibody (Bevacizumab (Avastin®)) toVK-B8, a disclosed herein fully human anti-VEGFR2 (receptor) antibody,for effects on MC38 colon cancer growth measured by solid tumor volume.C57BL6/6 mice were injected subcutaneously with 2×10⁶ MC38 colon tumorcells. After 4 days, a treatment protocol was initiated. Test antibodywas administered at 0.2 mg/mouse ip 3× per week in a volume of 0.1 ml.The tumor volume was measured as W×L×L/2 wherein W is tumor width and Lis tumor length. FIG. 8 shows that the disclosed antibody VK-B8 displaysgreater efficacy than the marketed antibody Bevacizumab (Avastin®;anti-VEGF ligand humanized antibody).

Example 4

This example illustrates in vivo data comparing a marketed anti-VEGF(ligand) humanized monoclonal antibody (Bevacizumab (Avastin®)) toVK-B8, a disclosed herein fully human anti-VEGFR2 (receptor) antibody,for effects on A431epidermoid carcinoma cell growth measured by solidtumor volume. Nude mice were injected subcutaneously with 2×10⁶ A431epidermoid carcinoma cells. After 4 days, a treatment protocol wasinitiated. Test antibody was administered at 0.2 mg/mouse ip 3× per weekin a volume of 0.1 ml. The tumor volume was measured as W×L×L/2 whereinW is tumor width and L is tumor length. FIG. 9 shows that the disclosedantibody, VK-B8, shows similar efficacy compared to the marketedantibody Bevacizumab (Avastin®; anti-VEGF ligand humanized antibody).

Example 5

This example illustrates in vivo data comparing a marketed anti-VEGF(ligand) humanized monoclonal antibody (Bevacizumab (Avastin®)) to (1)Erbitux (cetuximab), a humanized anti-EGFR antibody, (2) A6, a fullyhuman anti-EGFR antibody, (3) combination of A6 plus Bevacizumab(Avastin®), (4) combination of A6 plus Erbitux, and (5) A6 plus VK-B8wherein VK-B8 is a disclosed herein fully human anti-VEGFR2 (receptor)antibody for effects on A431epidermoid carcinoma cell growth bymeasuring solid tumor volume. Nude mice were injected subcutaneouslywith 2×10⁶ A431epidermoid carcinoma cells. After 4 days, a treatmentprotocol was initiated. Test antibody was administered at 0.2 mg/mouseip 3× per week in a volume of 0.1 ml. The tumor volume was measured asW×L×L/2 wherein W is tumor width and L is tumor length. FIG. 10 showssimilar efficacy for each of the treatment groups with the combinationof A6+Avastin® and A6+VK-B8 showing the highest efficacy for tumorgrowth inhibition.

Example 6

This example illustrates in vitro data for VK-B8 cellular EC₅₀measurements. Protocol: 50,000 HUVECs were aliquoted into the wells of a96-well, v-bottom plate in 100 ul FACS Buffer (PBS+2% FBS). A twelvepoint, 3× dilution curve of VK-B8 was made in FACS Buffer starting at 50μg/ml (3.33×10⁻⁷M). Cells were spun down, washed 1× with FACS Buffer,and then resuspended in 25 μl of antibody solution in triplicate. After0.5 hr. incubation, cells were washed 1× with FACS Buffer andresuspended in 50 μl PE-conjugated, goat anti-human IgG (γ-chainspecific) secondary antibody (Southern Biotech Cat #2040-09). Cells werefurther incubated for 0.5 hr. and then washed 1× with FACS Buffer. Cellswere resuspended in 25 μl FACS Buffer and the median fluorescenceintensity in the FL2-H channel was determined using the Intellicyt HTFCflow cytometer.

Results: The cell binding EC₅₀ for VK-B8 on HUVECs was determined to be1.3 nM. Data was analyzed and plotted in Graph Pad Prizm usingnon-linear regression fit. Data points are shown (FIG. 4) as the medianfluorescence intensity (MFI) of positively labeled cells+/−Std Error.

Example 7

This example illustrates in vitro data for cell binding as part of aninitial screen for several disclosed antibodies, including VK-B8.Protocol: 100,000 HUVECs were aliquoted into tubes in 100 μl FACS Buffer(PBS+2% FBS). Cells were spun down and then resuspended in 100 μl ofFACS Buffer plus 10 μg/ml of the indicated antibody in triplicate. After0.5 hr incubation, cells were washed 1× with FACS Buffer and resuspendedin 100 μl PE-conjugated, goat anti-human IgG (γ-chain specific)secondary antibody (Southern Biotech Cat #2040-09). Cells were furtherincubated for 0.5 hr and then washed 1× with FACS Buffer. Cells wereresuspended in 300 μl FACS Buffer and the median fluorescence intensityin the FL2-H channel was determined using the FACSAria flow cytometer(BD).

Results: The cell binding for the anti-VEGFR2 antibodies disclosed (FIG.3) on HUVECs was strong compared to background staining (control). Thehistograms shown depict the number events with specific fluorescenceintensities. Blue histograms are the VEGFR2-specific antibody while thered histograms are the background control. Data shown is representativeof multiple experiments.

Example 8

This example illustrates in vitro data showing inhibition ofVEGF-mediated HUVEC proliferation by VK-B8 (anti-VEGFR2) versus FDAapproved anti-VEGF Bevacizumab (Avastin®) Protocol: 5000 HUVECs wereplated into the wells of a 96-well white opaque cell culture cluster in100 μl EBM-2 media supplemented with the growth factors etc. (Lonza). 24hr later, media was removed, cells washed 1× with PBS, and then starvedfor 18 hr in 50 μl non-supplemented (basal) EBM-2 media. Antibodies werediluted to 2× the indicated concentration in 50 μl non-supplemented(basal) EBM-2 media then added to the cells after removal of thestarvation media. After 1 hr incubation, VEGF was added at aconcentration of 200 ng/ml in 50 μl (final concentration of VEGF is 100ng/ml). Cells were then incubated for 48 hr. after which the PromegaCell Titer Glo kit was used to evaluate proliferation. Luminescenceoutput is directly proportional to cell number.

Results: VK-B8 inhibited VEGF-stimulated HUVEC proliferation (FIG. 7).Proliferation inhibition was equal to that conferred by Bevacizumab(Avastin®) at the same dose. Data shown is the mean relative light unitsof triplicate samples+/−Std Error

Example 9

This example illustrates in vitro data showing VEGF stimulatedauto-phosphorylation of VEGFR2 receptor in HUVECs. Protocol: 100,000HUVECs were plated in the wells of a 12-well cell culture cluster in 2.5ml EBM-2 media supplemented with the growth factors etc. (Lonza). 24 hr.later, media were removed and the cells washed 1× with PBS, and thenstarved for 18 hr. in 1 ml non-supplemented (basal) EBM-2 media.Antibodies were diluted to 2× the indicated concentration in 1 mlnon-supplemented (basal) EBM-2 media then added to the cells afterremoval of starvation media. After 0.5 hr. incubation, VEGF was added ata concentration of 200 ng/ml in 1 ml basal media (final VEGFconcentration is 100 ng/ml). Cells were then incubated for 30 min. Cellswere washed with PBS and lysed in 1× Cell Lysis Buffer (Cell Signaling).Phosphorylation of VEGFR2 was detected using the Cell Signaling PathScan ELISA Antibody Pair (#7824) according to manufacturer's protocolwith modification of the volumes to support half-area plates (allvolumes cut in half).

Results: HUVECs were treated with 100 ng/ml VEGF to stimulate activatingauto-phosphorylation of VEGFR2. Pre-treatment of cells with VK-B8 andother clones blocked this activation of VEGFR2. Bevacizumab (Avastin®)treatment shows expected inhibition of VEGFR2 auto-phosphorylation. Datashown (FIG. 6) is the mean absorbance at 450 nm of triplicatesamples+/−Std Error.

Example 10

This example illustrates in vitro data showing the inhibition of VEGFstimulated auto-phosphorylation of the VEGFR2 receptor in HUVECs byVK-B8. Upon binding of VEGF, VEGFR2 undergoes activatingauto-phosphorylation at Tyrosine 1175 leading to downstream signalingevents and the activation of cellular events such as proliferation andcell migration. To assess the ability of VK-B8 to inhibit activation ofVEGFR2 by VEGF, 50,000 HUVECs were plated in the wells of a 96-well cellculture cluster in 100 μl EGM-2 media supplemented with growth factors(Lonza). 24 hr. later, the medium was removed and the cells washed 1×with PBS followed by starvation for 18 hr. in 100 μl non-supplemented(basal) EGM-2 media. Cells were then preincubated with a serial dilutioncurve of VK-B8 for 15 minutes, followed by incubation with VEGF at afinal concentration of 50 ng/ml for 5 minutes. Cells were washed withPBS and lysed in 1× Cell Lysis Buffer (Cell Signaling). Phosphorylationof VEGFR2 at Tyr1175 was detected using the Cell Signaling Path ScanELISA Antibody Pair (#7824) according to manufacturer's protocol withmodification of the volumes to support half-area plates (all volumes cutin half). Absorbance at 450 nm was plotted vs. antibody concentrationand non-linear regression was used to determine the IC₅₀ value (FIG.11). The IC₅₀ for VK-B8 inhibition of VEGF-induced VEGFR2auto-phosphorylation was 0.12 nM.

Example 11

This example illustrates in vitro data showing the inhibition of VEGFstimulated phosphorylation of p44/p42 MAPK (Erk1/2) by VK-B8 in HUVECs.Upon binding of VEGF, VEGFR2 undergoes activating auto-phosphorylation,establishing a signaling cascade which results in the activatingphosphorylation of p44/p42 MAPK (Erk1/2). To assess the ability of VK-B8to inhibit this activation, 50,000 HUVECs were plated in the wells of a96-well cell culture cluster in 100 ul EGM-2 media supplemented withgrowth factors (Lonza). 24 hr. later, the medium was removed and thecells washed 1× with PBS followed by starvation for 18 hr. in 100 μlnon-supplemented (basal) EBM-2 media. Cells were then preincubated witha serial dilution curve of VK-B8 for 15 minutes, followed by incubationwith VEGF at a final concentration of 50 ng/ml for 5 minutes. Cells werewashed with PBS and lysed in 1× Cell Lysis Buffer (Cell Signaling).Phosphorylation of p44/p42 MAPK (Erk1/2) was detected using a CellSignaling Path Scan ELISA Antibody Pair (#7246) according tomanufacturer's protocol with modification of the volumes to supporthalf-area plates (all volumes cut in half). Absorbance at 450 nm wasplotted vs. antibody concentration and non-linear regression was used todetermine the IC₅₀ value (FIG. 12). The IC₅₀ for VK-B8 inhibition ofVEGF-induced p44/p42 MAPK (Erk1/2) phosphorylation was 0.08 nM.

Example 12

This example illustrates in vitro data showing inhibition ofVEGF-mediated HUVEC proliferation by VK-B8. The effect of VEGFactivation of VEGFR2 on endothelial cells is to trigger proliferation.In oncology, a tumor will secrete VEGF to manipulate the vascularmicro-environment, leading to proliferation and expansion of thevasculature and finally invasion of the vasculature into the tumor.Inhibition of the process has been shown to functionally starve thetumor. To assess the inhibition of VEGF-induced endothelial cellproliferation by VK-B8, 5000 HUVECs were plated into the wells of a96-well cell culture cluster in 100 μl EGM-2 media supplemented withgrowth factors (Lonza). 24 hr later, media was removed, cells washed 1×with PBS, and then starved for 18 hr. in 100 μl non-supplemented (basal)EGM-2 media. Cells were then preincubated with a serial dilution curveof VK-B8 for 15 minutes, followed by incubation with VEGF at a finalconcentration of 50 ng/ml for 48 hr. To measure proliferation, thePromega Cell Titer 96 Non-Radioactive Cell Proliferation kit was used.Absorbance at 570 nm was directly proportional to cell number. Abs570 nmwas plotted vs. antibody concentration and non-linear regression wasused to determine the IC₅₀ for this effect (FIG. 13). The IC₅₀ value forthe inhibition of proliferation by VK-B8 was 14 nM.

Example 13

This example illustrates in vitro data showing inhibition ofVEGF-mediated HUVEC migration by VK-B8. An effect of VEGF activation ofVEGFR2 on endothelial cells is to trigger cell migration. In oncology, atumor will secrete VEGF to manipulate the vascular micro-environment,leading to migration and invasion of the vasculature into the tumor.Inhibition of this process has been shown to functionally starve thetumor. To assess the inhibition of VEGF-induced endothelial cellmigration by VK-B8, 80,000 HUVECs per sample were preincubated with aserial dilution curve of VK-B8 for 20 minutes and then plated onto thesites of the upper plate of a 96-well modified Boyden Chamber(NeuroProbe, 8 μm pores). The lower chamber of the plate contained VEGFat a final concentration of 5 ng/ml. Twenty hours after the plating ofcells, the number of cells which migrated through the membrane to thelower chamber were counted and plotted vs. the antibody concentration(FIG. 14). The IC₅₀ value for the inhibition of VEGF-induced HUVECmigration by VK-B8 was calculated using non-linear regression anddetermined to be 0.5 nM.

Example 14

This example illustrates in vitro data showing the inhibition of VEGF-Cstimulated auto-phosphorylation of the VEGFR2 receptor in HUVECs byVK-B8. In addition to VEGF (VEGF-A), other members of this growth factorfamily can bind and activate VEGFR2 leading to functional outcomes. Thegrowth factor VEGF-C can bind and activate VEGFR2 and VEGFR3. Here wedemonstrate the ability of VK-B8 to block the activation of VEGFR2 byVEGF-C. To assess the ability of VK-B8 to inhibit activation of VEGFR2by VEGF-C, 50,000 HUVECs were plated in the wells of a 96-well cellculture cluster in 100 μl EGM-2 media supplemented with growth factors(Lonza). 24 hr. later, the medium was removed and the cells washed 1×with PBS followed by starvation for 18 hr. in 100 μl non-supplemented(basal) EGM-2 media. Cells were then preincubated with a serial dilutioncurve of STI-A0168 (VK-B8) for 15 minutes, followed by incubation withVEGF-C at a final concentration of 1 μg/ml for 5 minutes. Cells werewashed with PBS and lysed in 1× Cell Lysis Buffer (Cell Signaling).Phosphorylation of VEGFR2 at Tyr1175 was detected using the CellSignaling Path Scan ELISA Antibody Pair (#7824) according tomanufacturer's protocol with modification of the volumes to supporthalf-area plates (all volumes cut in half). Absorbance at 450 nm wasplotted vs. antibody concentration and non-linear regression was used todetermine the IC₅₀ value (FIG. 15). The IC₅₀ for VK-B8 inhibition ofVEGF-C-induced VEGFR2 auto-phosphorylation was 1.9 nM.

Example 15

This example provides a summary table of affinity measurements, EC₅₀measurement, IC₅₀ measurements of the identified antibodies.

mAb VB-A2 VB-A9 VB-E1 VB-F2 VR-B2 RV-H5 VR-B4 VR-E3 ka 1.44E+6 3.71E+41.80E+5 6.16E+4 3.36E+5 5.39E+5 2.04E+5 3.75E+4 kd 1.55E−3 2.35E−31.62E−3 2.13E−3 3.19E−3 5.71E−3 2.54E−3 8.20E−4 Kd 1.08E−9 6.34E−89.02E−9 3.46E−8 9.50E−9 1.06E−8 1.25E−8 2.19E−8 EC50 0.50 0.25 0.5 weak0.747 0.323 24.3 1.29 nM IC50, 25.3 2.05 weak weak — 5.79 — 9.32 nMVK-A1 VK-B7 VK-B8 VK-C1 VK-E5 VK-G9 1121* ka 6.57E+4 7.56E+4 1.27E+51.71E+6 1.30E+5 1.10E+5 4.79E+5 kd 2.42 1.92E−3 7.75E−4 2.01E−3 1.16E−31.91E−3  2.5E−5 KD 3.55E−9 3.20E−8 6.11E−9 1.17E−8 8.97E−9 1.75E−8 5.0E−11 EC50 nM 0.109 1.89 0.25 2.0 0.175 0.653 0.25 IC50 nM 3.57 7.332.04 57.0 3.03 5.57 0.8 *published data in the literature: Lu, D., etal., 278: 43496-43507, JBC (2003)

Sequence Listing: Binder VH VL VB-A2 MAQVQLVQXGAEVKKPGASVKVSCKASGYTFTSYYAIQLTQSPSSLSASVGDRVTITCRASQGISNY MHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGLAWYQQKPGKVPKLLIYAASTLQSGVPSRFS RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRGGSGSGTDFTLTISSLQPEDFATYYCQQSYLTP MLRHWGMDVWGQGTTVTVSS WTFGQGTKVEIKSEQ ID NO. 1 SEQ ID NO. 2 VB-A3 MAQVQLVQSGAEVKKPGASVKVSCKASGHTFNYYYSYELTQPPSVSVAPGKTASITCGGNNIGSKS MHWVRQAPGQGLEWMGQTNLNSGGTNYAPKFQVHWYQQKPGQAPVLVIYYDRDRPSGIPERF GRVTMTRDTSISTAYMELSSLRSDDTAVYYCANLNSSGSNSGNTATLTISRVEAGDEADYYCQVWD GWFHFENWGQGTLVTVSS SSSYHPVFGGGTKLTVLSEQ ID NO. 3 SEQ ID NO. 4 VB-A5 MAQMQLVQSGAEVKKPGSSVKVSCEASGGTFSSFAQPVLTQPPSASGTPGQRVTIFCSGSTSNIGS ISWVRQAPGQGLEWMGRVIPVFGTANYAQTFQGNTVNWYHHLPGTAPKLLIYSNNQRPSGVPD RVTITADKSTSTMFMELSSLRSEDTAVYYCARESGDYRFSGSKSGTSASLAISGLQSADEADYYCAAW YDGSRYVDAFDIWGQGTMVTVSSDDNLNGWVFGGGTKLTVL SEQ ID NO. 5 SEQ ID NO. 6 VB-A7MAQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYY SYVLTQPASVSGSPGQSITISCAGTSSDIGGYMHWVRQAPGQGLQWMGIINPSGGSTSYAQNEQ NSVSWYQQNPGKAPKLMIYEGSKRPSGVSGRVTMTRDTSTSTVYMELSSLISQDTAVYYCARSGY NRFSGSKSGTTASLTISGLQAEDEADYYCSSYSSSWLSYGMDVWGQGTTVTVSS TNSDTWVFGGGTKLTVL SEQ ID NO. 7 SEQ ID NO. 8 VB-A9MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYS QSALTQPRSVSASPGQSVTISCTGTSSDVGGMNWVRQAPGKGLEWVSSISSGSSYIYYADSLKGRF YDYVSWYQQHPGKAPKLMIYEVSNRPSGVTISRDNAKNSLYLQMNSLRAEDTAVYYCARDFGNW SNRFSGSKSGNTASLSISGLQAEDEADYYCSGQGTLVTVSS SYTSTSSPVVFGGGTKLTVL SEQ ID NO. 9 SEQ ID NO. 10 VB-A10MAEVQLVESGGGLVQPGGSLRLSCAVSGFTLSSYE QPVLTQPPSASGTPGQRVTISCSGSSSNLGSMMWVRQAPGKGLEWVSYISDSGGLIYYSDSVKGR NYVYWYQHLPGTAPKLLIYRNKQRPSGVPDFTISRDSAKNSLYLQMNSLRDEDTAVYYCARDEYSS RFSGSKSGTSASLAISGLRSEDEADYYCAVWGMDVWGQGTTVTVSS DGSLSGYVFGTGTKLTVL SEQ ID NO. 11 SEQ ID NO. 12 VB-B6MAQVQLVQSGAEVKKPGASVKVSCKASGYTFSSYY QSVLTQPRSVSGSPGQSVTISCTGTSSDVGGMHWVRQAPGQGLEWMGIINPSAGSTNYAQKFQG YNYVSWYQQHPDKAPKLMLYDVSKRPSGVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYYY SNRFSGSKSGNTASLTIXALQAEDEADYYCSDTSGYHGYFKYWGQGTLVTVSS SYTSSTNLGVFGGGTKVTVL SEQ ID NO. 13 SEQ ID NO. 14VB-B10 MAEVQLLESGGGLVQPGRSLRLSCAASGFSFDDYAQSALTQPPSASGTPGQRVTISCSGSSSNIGT MHWVRQAPGKGLEWVSSISWNSGSRGYADSVKGNTVNWYQQLPGTAPRLLIYFNNQRPSGVP RFTISRDNAKNSLFLQMNSLRTEDTALYYCAKGVVYDRFSGSKSATSASLAISGLQSEDEADYYCSA SSPYYGMDVWGQGTTVTVSS WDDILNDPVFGGGTKLTVLSEQ ID NO. 15 SEQ ID NO. 16 VB-D5 MAQVQLVQSGAEVKKPGASVKISCKASGYIFNTYSIQAVLTQPPSASGTPGQRVTISCSGSSSNIGS HWVRQAPGQSFEWMGWSSAGDDNTKYSDDFHHNTVNWYQQLPGTAPKLLIYSNNQRPSGVP RLTIARDTSASTVYMELRGLTSDDTAIYYCARGYELDDRFSGSKSGTSASLAISGLQSEDEADYYCAA FWGQGTLVTVSS WDDSLNGDVVFGGGTKVTVLSEQ ID NO. 17 SEQ ID NO. 18 VB-D6 MAEVQLVESGGDLVKPGGSXRLSCAASGFSFSDYYQSALTQPASVSGSPGQTITISCAGTPSDIGLY MSWIRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTINYVAWFQHHPGKAPKLIIYDVTKRPSGTSN SRDNAKNSLYLQMNSLRAEDTAVYYCARDAPRYGRFSGSKSGNTASLTISGLQADDEADYFCSSY MDVWGQGTTVTVSS TTSNSFVLFGEGTKLTVLSEQ ID NO. 19 SEQ ID NO. 20 VB-D11 MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAQSVVTQQPSVSAAPGQKVTISCSGGSSNIG ISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRNNYVSWYQQLPGTAPKLLIYDNNKRPSGIP VTITADESTSTAYMELSSLRSEDTAVYYCARGSERVYDRFSGSKSGSSASLAITGLQAEDEADYYCQS SSHTYYGMDVWGQGTTVTVSSYDSSLSGYVFGTGTKLTVL SEQ ID NO. 21 SEQ ID NO. 22 VB-E1MAQVQLVESGAEVKKPGASVKVSCKASGYTFTNYYI DIVMTQSPDSLAVSLGERATINCKTSQSVLYHWVRQAPGQGLEWMGIINPSSGSTNYAQKFQGR NANNKNYLNWYQQKPGQPPKLLIYWASARVTMTRDTSTSTVYMELSSLRSEDTAVYYCARQRWD ESGVPDRFSGSGSGTDFTLTIRSLQPDDFATLLDDAFDIWGQGTMVTVSS YYCQQAISFPLTFGGGTKVEIK SEQ ID NO. 23 SEQ ID NO. 24VB-E2 MAEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYG SYELMQPPSVSEAPGMTAQITCGGNNIGSKMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKG SVHWYQQKPGQAPVLVIYYDSERPSGIPDRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDWS FSGSNSGNTASLTINRVEAGDEADYYCQVWGPITLWGQGTLVTVSS DSSSDHHVVFGGGTKLTVL SEQ ID NO. 25 SEQ ID NO. 26 VB-E7MAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYE QSALTQPPSMSAAPGQKVTISCSGTSSNIGMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRF NHYVSWYQRLPDTAPKLLIYDNNRRPSGIPTISRDNAKNSLYLQMNGLRAEDTAVYYCAREGDSS DRFSGSKSGTSATLGITGLQTGDEADYYCGTGFDYWGQGTLVTVSS WDSSLSGYVFGTGTKVTVL SEQ ID NO. 27 SEQ ID NO. 28 VB-F2MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSDHY DIVMTQTPLFLPVTLGQPASISCRSSQSLVHSMDWVRQAPGKGLEWVGRIRNKPNSYTSEYAASVK DGNTYLNWFQQRPGQSPRRLIYKVSIRDSGGRFTISRDDSKNSLYLQMNSLRAEDTAVYYCASRSG VPGRFSGSGSGTDFTLKISSVEAEDIGVYYCSYYDHMDVWGQGTTVTVSS MQGTDRPYTFGQGTKLEIK SEQ ID NO. 29 SEQ ID NO. 30VB-F8 MAQMQLVQSGGGVVQPGRSLRLSCVVSGFTFNDY QAGLTQSPSVSAAPGQRVTISCTGSSSNIGAPMHWVRQAPGKGLEWVALLSYDGTSAYYADSVEG GYDVHWYQQLPGTAPKLLIYGNSNRPSGVPRFTISRDNSKNTLYLQMNTLRTEDTAVYYCASEGSP DRFSGSKSGTSASLAITGLQAEDEADYYCQSDAFDIWGQGTMVTVSS YDSSLSGSVFGGGTKVTVL SEQ ID NO. 31 SEQ ID NO. 32 VB-G4MAQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI QSVVTQPASVSGSPGQSITISCTGTTTDVGAGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ YNYVSWYQHHPGKAPKLIIYDLNNRPSGISNVTISADKSISTAYLQWSSLKASDTAMYYCARLDYDSS RFSGFKSGNTASLTISGLQAEDEADYYCSSYTGYYGAFDIWGQGTMVTVSS SSSTWVFGGGTKLTVL SEQ ID NO. 33 SEQ ID NO. 34 VB-G6MAQVQLVESGAEVKKPGASVKVSCKASGYTFTSYY QAGLTQPPSASGSPGQSVTISCTGTSSDVGMHWVRQAPGQGLQWMGIINPSGGSTSYAQNEQ GYNYVSWYQQHPGKAPKLMIYEVSKRPSGGRVTMTRDTSTSTVYMELSSLISEDTAVYYCARSGYS VPDRFSGSKSGNTASLTVSGLQAEDEADYYSSWLSYGMDVWGQGTTVTVSS CSSFTTSSTWVFGGGTQLTVL SEQ ID NO. 35 SEQ ID NO. 36VB-H4 MAEVQLVQSGAEVKKPGASVKLSCKASGYTFNNYAQPVLTQPPSASGTPGQRVTIYCSGSNSNIGG TIWVRQAPEQGLEYVGWISAYSGHTNYAQKLQGRNSVNWYQQLPGTAPKLLIYHNNQRPSGVP VSMTTDTSTTTAYMELRSLRSDDTAVYYCARWSGDRFSGSRSGTSASLAISGLQSGDEADYSCAA WGSYHLLGMDVWGQGTTVTVSSWDDSLRGYVFGTGTKVTVL SEQ ID NO. 37 SEQ ID NO. 38 VB-H7MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYY QAVLTQPASVSGSPGQSITISCTGTSSDVGGMHWVRQATGQGLEWLGWMNPKTGVTGYAQKF YNYVSWYQQHPGKAPKLMIYEVNNRPSGVQGRVTMTRNTSINTAYMELNSLTSEDTADYYCARG SNRFSGSKSGNTASLTISGLQAEDEADYYCTDYGGPQDMDVWGQGTTVTVSS SYTSSNTRMFGGGTKLTVL SEQ ID NO. 39 SEQ ID NO. 40VB-H9 MAEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYQAVLTQPRSVSGSPGQSVTISCTGTRSDVGT MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFYNYVSWYQQLPGKAPKLMIHDVSKRPSGV QGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLPDRLSGSKSGNTASLTISGLQSEDEADYYCIS QGITIFGVANSPPYYYGMDVWGQGTTVTVSSYTLHRTWMFGGGTKLTVL SEQ ID NO. 41 SEQ ID NO. 42 RV-A9MAEVQLVQSGAEVKKPGASVKISCKASGYIFNTYSIH QPVLTQPPSVSGTPGQRVSISCSGSSSNIGSWVRQAPGQSFEWMGWSSAGDDNTKYSDDFHHR NSVNWYQQLPGTAPRLLIYNNDQRPSGVPLTIARDTSASTVYMELRGLTSDDTAIYYCARGYELDF DRFSASKSGTSASLAIGGLQSEDEGDYYCSAWGQGTLVTVSS WDDSLNGPWVFGGGTKLTVL SEQ ID NO. 43 SEQ ID NO. 44 RV-F8MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYS QSVLTQPPSVSAAPGQKVTISCSGSTSNIANMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFT NFVSWYQQLPGTAPKLLIYDNNKRPSGIPDISRDNAKNSLYLQMNSLRAEDTAVYYCARVGATMG RFSGSKSGTSATLGITGLQAGDEADYFCGTDYWGQGTLVTVSS WDSSLSASYVFGTGTKVTVL SEQ ID NO. 45 SEQ ID NO. 46 RV-H2MAQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI DIVMTQSPSSVSAFVGDRVTITCRASQDVGGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ SWLVWYQQKPGKVPKLLIYGASTLQSGVPSVTISADKSTSTAYLQWSSLKASDTAMYYCARLGSSG RFSGGGSGTDFTLTISSLQPEDFATYYCQQAWYDAFDIWGQGTMVTVSS KSLPYTFGQGTKLEIK SEQ ID NO. 47 SEQ ID NO. 48 RV-H4MAQVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYS LPVLTQPASVSGSPGQSITISCTGTDSDVGGMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRET YNYVSWYQQHPGKAPKLIISDVSNRPSGVSISRDNAKNSLYLQMNSLRAEDTAVYYCARVGATMG NRFSGSKSGNTASLTISGLQADDEADYYCNSDYWGQGTLVTVSS YTVHATVLFGGGTKVTVL SEQ ID NO. 49 SEQ ID NO. 50 RV-H5MAEVQLLESGGGLVKPRGSLRLSCAASGFTFSNAW QAGLTQPPSVSVSPGQTASITCSGDKLGDKYMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVK ISWYQQKPGQSPVMVIFQDTKRPSGIPERFGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTVWR SGSNSGNTATLTISGTQAMDEADYYCQAWFGELFRWGQGTLVTVSS DTSTVFGGGTKLTVL SEQ ID NO. 51 SEQ ID NO. 52 C1MAEVQLVQSGSELKKPGASVKISCKASGYTLTNHAL QPVLTQPPSVSVAPGQTARITCGGNNIGSKNWVRQAPGQGLEWMAWMNTNTGNPTYAQDFT LVHWYQQKPGQAPVLVVYDDSDRPSGIPEGRFVFSLDTSVSTAYLEISSLKAEDTAIYYCAREPRDA RFSGSNSGNTATLTISRVEAGDEADYYCQVDAFDIWGQGTMVTVSS WDSSSDLVVFGGGTKLTVL SEQ ID NO. 53 SEQ ID NO. 54 VR-A2MAQVQLVQSGTEVKKPGESLRISCRSSGYKFTNYWI QSVVTQPPSVSAAPRQKVTISCSGSSSNIGNGWVRQLPGQGLEWMGVILPGDSDTRYGPSFQGH NYVSWYQQLPGTAPKLLIYDNNRRPSGIPDVSISVDKSISTVYLEWESLKASDTAMYYCASWDNFD RFSGSKSGTSATLGITGLQTGDEADYYCGTHWGQGTLVTVSS WDSSLSAGVFGTGTKVTVL SEQ ID NO. 55 SEQ ID NO. 56 VR-A3MAEVQLVQSGAEVKKPGTSVTISCKTSGYTFTTYYIH AIRMTQSPDSLAVSLGERATINCKSSQSVLYWVRQAPGQGLEWMGIILPSGGNTNYAPNFQGRV SSNNKNYLAWYQQKPGQPPKLLIYWASTRETMTRDTSTSTVNMELSSLTSDDTAVYYCVREYRGGY SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFDYWGQGTLVTVSS YCQQYYSTPYTFGQGTKLEIK SEQ ID NO. 57 SEQ ID NO. 58 VR-A10MAQVQLVESGAEVKKPGASVKVSCKASGYTFTSYY QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQG GYDVHWYKQLPGTAPKLLIYGNNNRPSGVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGAN PDRFSGSKSGTSASLAITGLQAEDEADYYCQAPNSYYYMDVWGKGTTVTVSS SYDSSLSEGVFGTGTKVTVL SEQ ID NO. 59 SEQ ID NO. 60VR-B2 MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYADIQLTQSPPSVSASVGDRVTITCRASQDIST MHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGWLAWYQQKPGSAPKVLIYAASTFQSGVPSR RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLGAFRGSGSGTYFTLTISGLQPEDFATYYCQQGN ASYWYFDLWGRGTLVTVSS SFPPTFGQGTKLEIKSEQ ID NO. 61 SEQ ID NO. 62 VR-B4 MAQVQLVQSGAEVKKPGSSVKVSCKAYGGTFGSYSYELTQPASVSGSPGQSITISCTGSSSDVGGY GVSWVRRAPGQGLEWMGRLIPIFGTRDYAQKFQGNFVSWYRQHAGKAPKLMIYDVTNRPSGVS RVTLTADESTNTAYMELSSLRSEDTAVYYCARDGDYTRFSGSKSGTTASLTISGLQPDDEAHYYCSSY YGSGSYYGMDVWGQGTTVTVSSTTTSTWVFGGGTKLTVL SEQ ID NO. 63 SEQ ID NO. 64 VR-B11MAEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYG QSVVTQPPSVSAAPGQRVTISCTGSSSNIGAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKG GYDVHWYQQLPGTAPKLLIYANNNRPSGVRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGYS PDRFSGSKTGTSASLAITGLQADDEADYFCQYGGGFDYWGQGTLVTVSS SYDSSLSGWVFGGGTKLTVL SEQ ID NO. 65 SEQ ID NO. 66VR-C5 MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSQSVLTQPASVSGSPGQSITISCTGTSSDVGG MNWVRQAPGKGLEWVSSISSGSSYIYYADSLKGRFYNYVSWYQQHPGKAPKLMIYDVSNRPSGV TISRDNAKNSLYLQMNSLRAEDTAVYYCARDFGNWSNRFSGSKSGNTASLTISGLQAEDEADYYCS GQGTLVTVSS SYTSSSTPYVFGTGTKVTVLSEQ ID NO. 67 SEQ ID NO. 68 VR-C7 MAQVQLVESGGGLVQPGGSLRLSCAASGFNFSSYESYELMQPHSVSESPGKTVTISCTGSSGSIASN MNWVRQAPGKGLEWVSYISSSGSTKHYADSVKGRYVQWYQQRPGSAPTTVIYEDDQRPSGVPD FTISRDNAKNSLYLQMNSLRAEDTAVYYCAREHYNSRFSGSIDSSSNSAALTISGLKTEDEADYYCQS WYFDLWGRGTLVTVSS YDSANVVFGGGTKLTVLSEQ ID NO. 69 SEQ ID NO. 70 VR-C11 MAEVQLLESGGGWVKPGGSLRLSCAASGFPFSDYYLPVLTQPPSVSAAPGQKVTIPCSGTYSNIVN MTWVRQAPGKGLEWVSYITTGGRIlYSADSVRGRENYVSWYQQLPGTAPKLLIYDNNKRPSGIPD TISRDNAKNSVYLQMNSLRAEDTAVYYCARPLRELSRFSGSKSGTSATLGITGLQTGDEADYYCGT PGGFDLWGRGTMVTVSS WDNSLRRWVFGEGTKLTVLSEQ ID NO. 71 SEQ ID NO. 72 VR-E3 MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSQPVLTQPPSASGSPGQSVTISCTGTSSDVGG MNWVRQAPGKGLEWVSSISSASSYIYYADSVKGRFYNYVSWYQQHPGKAPKLMIYDVNNRPSGV TISRDNAKKSLYLQLNSLTVEDTAVYYCAREYWGSPPDRFSGSKSGNTASLTISGLQAEDEADYYCS DYWGRGTLVTVSS SYTSSSTRVFGGGTKLTVLSEQ ID NO. 73 SEQ ID NO. 74 VR-G11 MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSQSVVTQPPSVSAAPGQKVTISCSGSSSNIGN MNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTNYVSWYQQLPGTAPKLLIYDYNKRPSGIPDR ISRDNAKNSLYLQMNSLRAEDTAVYYCARGGVSPGFSGSQSGTSATLGITGLQTGDEADYYCGTW DYWGQGTLVTVSS DSSLTLYVFGTGTKLTVLSEQ ID NO. 75 SEQ ID NO. 76 VK-B8 MAQVQLVQSGAEVKKPGSSVKVSCKAYGETTLTQSPATLSVSPGERATVSCRASQSLGS GTFGSYGVSWVRRAPGQGLEWMGRLIPINLGWFQQKPGQAPRLLIYGASTRATGIPAR FGTRDYAQKFQGRVTLTADESTNTAYMELFSGSGSGTEFTLTISSLQSEDFAVYFCQQYN SSLRSEDTAVYYCARDGDYYGSGSYYGMDDWPITFGQGTRLEIK VWGQGTLVTVSS SEQ ID NO. 78 SEQ ID NO. 77 VR-H9MAQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSY QPVLTQPPSVSKDLRQTATLTCTGNGNNVGAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGR YQGAAWLQQHQGHPPKLLSYRNNNRPSGIVTITADKSTSTAYMELSSLRSEDTAVYYCARGHDYYG SERFSASRSGNTASLTISGLQPEDEADYFCSASGNNQEDYFDPWGQGTLVTVSS WDNSLSAWVFGGGTKLTVL SEQ ID NO. 79 SEQ ID NO. 80VK-B8A MAQVQLVQSGAEVKKPGSSVKVSCKAYG ETTLTQSPATLSVSPGERATVSCRASQSLGSGTFGSYGVSWVRRAPGQGLEWMGRLIPI NLGWFQQKPGQAPRLLIYGASTRATGIPARFGTRDYAQKFQGRVTLTADESTNTAYMEL FSGSGSGTEFTLTISSLQSEDFAVYFCQQYNSSLRSEDTAVYYCARDGDYYGSGSYYGMD DWPITFGQGTKLEIK VWGQGTLVTVSS SEQ ID NO. 81SEQ ID NO. 77

1.-12. (canceled)
 13. An antigen-binding protein that binds to VEGFR2,comprising a heavy chain variable domain and a light chain variabledomain, wherein the heavy chain variable domain comprises CDRs as setforth in SEQ ID NO. 67 and the light chain variable domain comprisesCDRs as set forth in SEQ ID NO.
 68. 14. The antigen-binding protein ofclaim 13, wherein the heavy chain variable domain sequence has at least95% identity to the sequence of SEQ ID NO. 67, and the light chainvariable domain sequence has at least 95% identity to the sequence ofSEQ ID NO.
 68. 15. The antigen-binding protein of claim 13, wherein theheavy chain variable domain sequence comprises the sequence of SEQ IDNO. 67, and the light chain variable domain comprises the sequence ofSEQ ID NO.
 68. 16. The antigen-binding protein of claim 15, which is afully human antibody of an IgG class.
 17. The antigen-binding protein ofclaim 15, which is a Fab fully human antibody fragment.
 18. Theantigen-binding protein of claim 15, which is a single chain humanantibody, further comprising a peptide linker connecting the heavy chainvariable domain and the light chain variable domain.
 19. A method oftreating a VEGFR2-related disorder or condition in a human subject inneed thereof, the method comprising administering an effective amount ofthe antigen binding protein, the antibody, the antibody Fab fragment orthe single-chain antibody of claim 13 to the subject.
 20. The method ofclaim 19, wherein the VEGFR2-related disorder or condition is cancer, abenign tumor, an inflammatory disorder, or an ocular angiogenic disease.21. The method of claim 19, wherein the VEGFR2-related disorder orcondition is a solid tumor, a blood borne tumor, or a tumor metastasis.22. The method of claim 19, wherein the VEGFR2-related disorder orcondition is a hemangioma, an acoustic neuroma, a neurofibroma, atrachoma, or a pyogenic granuloma.
 23. The method of claim 19, whereinthe VEGFR2-related disorder or condition is chronic articularrheumatism, psoriasis, diabetic retinopathy, retinopathy of prematurity,macular degeneration, corneal graft rejection, neovascular glaucoma,retrolental fibroplasia, rubeosis, Osler-Webber Syndrome, myocardialangiogenesis, plaque neovascularization, telangiectasia, a hemophiliacjoint, angiofibroma, wound granulation, wound healing, telangiectasiapsoriasis scleroderma, pyogenic granuloma, coronary collaterals,ischemic limb angiogenesis, a corneal disease, rubeosis, arthritis,diabetic neovascularization, a fracture, or vasculogenesis.
 24. Apharmaceutical composition comprising the antigen-binding protein ofclaim 13, and a pharmaceutically acceptable carrier.
 25. A method oftreating a VEGFR2-related disorder or condition in a human subject inneed thereof, the method comprising administering an effective amount ofan anti-VEGFR2 antigen-binding protein to the subject, wherein theanti-VEGFR2 antigen-binding protein a fully human antibody of an IgGclass, a Fab fully human antibody fragment, or a single chain humanantibody; wherein the antigen-binding protein comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises the sequence of SEQ ID NO. 67 and thelight chain variable domain comprises the sequence of SEQ ID NO.
 68. 26.The method of claim 25, wherein the anti-PD-L1 polypeptide is the fullyhuman antibody.
 27. The method of claim 25, wherein the anti-PD-L1polypeptide is the Fab fully human antibody fragment.
 28. The method ofclaim 25, wherein the anti-PD-L1 polypeptide is the single chain humanantibody.
 29. The method of claim 25, wherein the mammalian cancer isovarian, colon, breast, lung cancers, myelomas, neuroblastic-derived CNStumors, monocytic leukemias, B-cell derived leukemias, T-cell derivedleukemias, B-cell derived lymphomas, T-cell derived lymphomas, mast cellderived tumors, or combinations thereof.
 30. The method of claim 25,wherein the autoimmune disease or inflammatory disease is intestinalmucosal inflammation, wasting disease associated with colitis, multiplesclerosis, systemic lupus erythematosus, viral infections, rheumatoidarthritis, osteoarthritis, psoriasis, Cohn's disease, and inflammatorybowel disease.
 31. A nucleic acid encoding the antigen-binding proteinof claim
 13. 32. A host cell comprising the nucleic acid of claim 31.33. A method of expressing the antigen-binding protein of claim 13,comprising culturing a host cell comprising a nucleic acid encoding theantigen-binding protein such that the antigen-binding protein isexpressed.
 34. The method of claim 33, further comprising isolating theantigen-binding protein.